Mica binding agents

ABSTRACT

The present invention relates to methods for the treatment of disorders mediated by MICA-expressing cells using antibodies, antibody fragments, and derivatives thereof that specifically bind MICA. The invention also relates to antibodies; cells producing such antibodies; methods of making such antibodies; fragments, variants, and derivatives of the antibodies; and pharmaceutical compositions comprising the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.61/595,902, filed 7 Feb. 2012 and 61/625,841, filed 18 Apr. 2012; all ofwhich are incorporated herein by reference in their entirety; includingany drawings.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled“PCT Seq list MICA_ST25”, created 7 Feb. 2013, which is 77 KB in size.The information in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides antigen-binding proteins capable ofbinding to MICA polypeptides. The antigen-binding proteins haveincreased activity in the treatment of disorders characterized byMICA-expressing cells, particularly tumor cells.

BACKGROUND

The immunoreceptor NKG2D is normally expressed on human T cells (e.g.CD8⁺ T cells, γδ T cells) and NK cells. On pre-activated CD8⁺ cells,NKG2D functions as a synergistic co-stimulator of CD28 and TCRsignalling via DAP10 association, whereas in NK cells it functions as adirect activator. Upon ligand engagement, NKG2D therefore conveysdirectly activating or costimulatory signals via the paired DAP10adaptor protein, thereby promoting cancer and infectious diseaseimmunity.

Various ligands for human NKG2D (hNKG2D) have been identified andcharacterized, including the major histocompatibility complex classI-related chain A and B polypeptides (MICA and MICB), the UL16-bindingprotein (ULBP) family, and the retinoic acid early transcript-1 (RAET1)family. MICA is frequently associated with epithelial tumors, induced bymicrobial infections, and aberrantly expressed in certain autoimmunedisease lesions. The structure of MICA is similar to the protein fold ofMHC class I, with an α 1α2 platform domain and a membrane-proximalIg-like α3 domain (Li et al 2001 Nat. Immunol. 2:443). MICA and itsclose relative MICB, which also serves as a ligand for NKG2D, are bothpolymorphic and the polymorphism has been shown to affect the affinityfor NKG2D (Steinle et al. 2001 Immunogenetics 53:279).

In the mouse, which lacks MHC class I chain (MIC) genes, a family ofproteins structurally related to ULBP, the retinoic acid early (RAE-1)molecules function as ligands for NKG2D. RAE-1 expression has been shownto be induced by carcinogens and to stimulate antitumor activities of Tcells. Murine NKG2D, however, recognizes human MICA polypeptides(Wiemann (2005) J. Immunol. 175:820-829).

The role MICA in cancer biology has been complicated by the fact thatMICA is released as a soluble form from the cell surface of tumor cells(e.g., *019 allele) and on the surface of exosomes (*08 allele) (Ashiruet al (2010) Cancer Res. 70(2):481-489)). Soluble MICA (sMICA) can bedetected for example at high levels in sera of patients withgastrointestinal malignancies (Salih et al, 2002 J. Immunol. 169: 4098).The MMPs ADAM10 and ADAM17, as well as the disulfide isomerase Erp5,have been reported to have a role in cleavage and shedding of MICA(Waldhauer (2008) Cancer Research 68 (15) 6368-76; Kaiser et al (2007)Nature; and Salih (2002) J. Immunol 169: 4098-4102). Membrane bound MICAhas been reported to downmodulate the expression of NKG2D on NK and/or Tcells (Von Lilienfeld-Toal et al. (2010) Cancer Immunol. Immunother.).Notably, Wiemann (2005), supra, examined MICA Tg mice and concluded thatdownregulation of surface NKG2D on nontransgenic splenocytes was mostpronounced after cocultivation with splenocytes from MICA transgenicmice in vitro, and only marginally following treatment with sera fromH2Kb-MICA mice, whereas incubation with control cells and sera fromnontgLM, respectively, had no effect and that overall data suggest thatreduced surface NKG2D on H2-K-MICA NK cells results in NKG2D dysfunctionand that NKG2D downregulation is primarily caused by a persistentexposure to cellbound MICA in vivo.

Reports have also emerged that NKG2D on NK cells is downregulated bysMICA (Groh et al. (2002) Nature; Arreygue-Garcia (2008) BMC; Jinushi etal. (2005) J. Hepatol.), leading to less reactive NK cells. Thisrationale may have emerged because similar systems have been observedamong other protein families such as the Ig-like and the TNF superfamilyhave been shown to be released as a soluble form and that release of themolecules affects cell-cell interactions by reduction of liganddensities and modulates NK cells bearing the respective receptor (Salih2002). Consequently, attempts to generate anti-MICA antibodies havefocused on development of antibodies that inhibit MICA shedding.

It has also been reported that expression of NKG2D ligands MICA and MICBon healthy cells can break the balance between immune activation andtolerance, and trigger autoimmunity. Genetic linkage studies have shownthat some MICA alleles are positively associated with type 1 diabetes,and development of disease in prediabetic NOD mice expressing Rae1 ontheir islet cells can be completely prevented by treatment withNKG2D-blocking mAbs, which reduce expansion and function of autoreactiveCD8+ T cells. MICA and MICB molecules are also dramatically upregulatedin RA synoviocytes and activate the T cells in an NKG2D-dependentmanner. Moreover, rheumatoid arthritis patients have been reported tohave high levels of IL-15 and TNF-α in the sera and inflamed jointswhich induce expression of NKG2D on CD4+CD28− subset of T cells. InCeliac disease, massive infiltration of intraepithelial NKG2D+CD8+ cd Tlymphocytes in the gut has been reported, and MIC proteins becomestrongly expressed on the surface of epithelial cells in patients withactive disease. In inflammatory bowel disorders, increased levels of MICexpression were found on intestinal epithelial cells and it the numberof intestinal epithelial CD4+ T cells expressing NKG2D was found tocorrelate with intestinal inflammation.

Approaches to date to treat inflammation based on the NKG2D system havefocused on blockade of NKG2D itself rather than its ligands (Ogasawaraet al. (2004) Immunity 20(6):757-767; Andersson et al (2011) Arthritis.Rheum. 63(9):2617-2629; Steigerwald et al (2009) MAbs 1(2):115-127. Onepossibility is that this focus on NKG2D rather than its ligand is due tothe perceived difficulty of targeting the NKG2D ligand system whichincludes a variety of ligands and in some cases a large number ofalleles.

For MICA and MICB, there are over 50 MICA alleles and at least 13 MICBalleles recognized. There is only 43% amino acid identity across the MICpolypeptides in the α1α2 domain (the domain involved in the NKG2Dinterface), and 80% of the amino acid substitutions are non-conservative(Steinle et al. (2001) Immunogenetics 53: 279-287; Steinle et al. (1998)Proc. Natl. Acad. Sci. U.S.A. 95:12510-12515), suggesting that it willbe unlikely to obtain antibodies that are effective for a majority ofindividuals in a population. Additionally, the methionine/valinebimorphism at position 129 in MICA determines differences in NKG2Dbinding, and although the side chain of residue 129 is partially buriedand forms hydrophobic interactions with glutamine 136, alanine 139 andmethionine 140 in the first α2 helical stretch, it may be associatedwith a difference in conformation in this domain in comparison withvaline 129 forms of MICA (Steinle et al (2001) Immunogenetics 53:279-287).

In conclusion, there is a need for new approaches to target MICA withtherapeutic agents.

SUMMARY OF THE INVENTION

In one aspect, the invention results, inter alia, from the discovery ofantibodies with high affinity across human MICA alleles (as well as onnon-human primate MICA).

The antibodies notably bind one or more MICA alleles from each of twomajor MICA groups that are determined to represent the main families ofMICA: Group 1 alleles that bind NKG2D strongly (including MICA*001,*002, *007, *012, *017 and *018) and Group 2 that bind NKG2D weakly(MICA*004, *006, *008, *009 and *019). By binding to an epitope presenton the subset MICA *001, *004, *007 and *008 or *001, *004, *007, *008and *019, the antibodies cover the alleles of both groups that arepresent in almost all individuals. Optionally, the antibodies have anEC50 of no more than 5 μg/ml, optionally no more than 3 μg/ml, no morethan 2 μg/ml, no more than 1 μg/ml or no more than 0.5 μg/ml for bindingto cells made to express at their surface *001, to cells made to expressat their surface *004, to cells made to express at their surface *007and to cells made to express at their surface *008.

High affinity binding is advantageous, inter alia, for an antibody toeffectively mediate CDC and ADCC. The invention provides epitopes onMICA within the α1 and/or α2 domains that are optimal antibody bindingregions for inducing high ADCC and/or CDC activity yet still are foundacross principal MICA alleles. The epitopes are generally on the lateralside of the α1 and/or α2 domains and are either entirely outside theNKG2D binding surface or partly overlapping with the NKG2D bindingsurface. Additionally, α3 epitopes are identified that exhibit enhancedADCC/CDC and multiple-allele binding characteristics.

Subgroups of antibodies were also identified that block the interactionof MICA with NKG2D. In addition to induction of ADCC and CDC activitywhen comprising Fc domains that are bound by Fcγ receptors or blockadeof pro-inflammatory activity when comprising Fc domains that are notsubstantially bound by Fcγ receptors, these antibodies were useful fortheir ability to be able to block sMICA-induced downmodulation of NKG2D.

Other subgroups of antibodies were also identified that did not blockthe ability of MICA on the surface of cells (e.g. tumor cells,transfectands) to induce NKG2D activity in a NKG2D-expressing immunecell that is brought into contact with said MICA-expressing cell in thepresence of anti-MICA antibody. In addition to induction of ADCC and CDCactivity, these antibodies were useful for their ability to avoidinhibition of NKG2D such that NKG2D-expression immune effector cellsremain able to lyse target cells via NKG2D (e.g. in addition to any Fcγreceptor-mediated mechanism).

Recombinant and cell surface bound MICA appear to be capable ofdifferent conformations and binding cell-bound MICA may have distanteffects on the MICA protein. Particularly surprisingly, blockade of theability of MICA on the surface of cells (e.g. tumor cells,transfectants) to induce signaling by NKG2D did not always correlatewith ability to block MICA-NKG2D interactions when recombinant proteinswere used (Biacore studie). Also, particularly surprisingly, whilepan-allele antibodies were not found completely within the NKG2D bindingzone on the plateau of the α1α2 domain, MICA blockade of the ability ofMICA on the surface of cells (e.g. tumor cells, transfectands) to inducesignaling by NKG2D did not correlate with the location of the bindingepitope. Some antibodies located far from the NKG2D interaction areawere able to block induction of NKG2D activity while some antibodiesnear the NKG2D binding area or with partial overlap did not blockinduction of NKG2D activity. Antibodies furthermore differed in theability to mediate CDC as a function of their epitopes.

Group 1 alleles of MICA generally have M at residue 129 while Group 2alleles have V at residue 129. In one embodiment, MICA groups arecharacterized by the presence of a methionine (M) or valine (V) residueat position 129 of the MICA polypeptide, wherein M is associated with aMICA form that binds strongly to NKG2D and V is associated with lowerbinding to NKG2D.

In one embodiment, the invention provides antibodies that cross-reactwith a MICA allele having a methionine at position 129 and a MICA allelehaving a valine at position 129. In one aspect the invention provides amonoclonal antibody that specifically binds to a human MICA polypeptidehaving a methionine at position 129 and a human MICA polypeptide havinga valine at position 129. Optionally, the antibodies have an EC50 of nomore than 5 μg/ml, optionally no more than 3 μg/ml, no more than 2μg/ml, no more than 1 μg/ml or no more than 0.5 μg/ml for binding tocells made to express at their surface a human MICA polypeptide having amethionine at position 129 and to cells made to express at their surfacea human MICA polypeptide having a valine at position 129.

In one embodiment the antibody further binds to a MICB polypeptidehaving a valine at position 152 (e.g., to a MICB polypeptide of SEQ IDNO: 6).

The binding regions discovered remain present on glycosylated MICA,notably MICA with glycosylation expressed preferentially by human tumorcells.

In one embodiment, the present invention results, inter alia, from thediscovery of antibodies that are effective in vitro and in vivo ininducing effector cell lysis (e.g. NK cells and/or T cells) ofMICA-expressing tumor cells while blocking the interaction of MICA withNKG2D. Antibodies that block NKG2D-MICA interactions be advantageous inthat such antibodies may prevent the sMICA-induced downregulation ofNKG2D as shown herein. Such blocking antibodies may be particular usefulfor the treatment of patients having high levels of soluble MICA, e.g.in circulation. In another embodiment, the present invention providesantibodies that are effective in vitro and in vivo in inducing effectorcell lysis (e.g. NK cells and/or T cells) of MICA-expressing tumor cellsand that inhibit sMICA-induced downmodulation of NKG2D expression on thesurface of an immune effector cell without substantially blockingshedding of MICA from MICA-expressing cells (e.g. tumor cells).

Such antibodies can inhibit sMICA-induced downmodulation of NKG2Dexpression by inhibiting the interaction of MICA with NKG2D. Optionallythe antibody comprises the light and heavy chain CDRs, optionally withone or more amino acid modifications in a CDR, of 9C10, 19E9, 12A10,18E8, 14B4 or 10F3.

In another embodiment, the present invention provides antibodies thatare effective in vitro and in vivo in inducing effector cell lysis (e.g.NK cells and/or T cells) of MICA-expressing tumor cells withoutsubstantially blocking shedding of MICA from MICA-expressing cells (e.g.tumor cells) and without substantially blocking the interaction of MICAwith NKG2D. Optionally the antibody comprises the light and heavy chainCDRs, optionally with one or more amino acid modifications in a CDR, of6E4, 20C6, 16A8, 15F9 10A7 or 14B4.

In one embodiment, antibodies that bind to the α1α2 domain (the domaininvolved in the NKG2D interface) are provided that cross-react withmultiple MICA alleles (e.g. a MICA allele having a methionine atposition 129 and a MICA allele having a valine at position 129; a MICA*001, *004, *007 and *008 allele) and bind with high affinity to suchMICA alleles (e.g. an EC50 of no more than 5 μg/ml, optionally no morethan 3 μg/ml, no more than 2 μg/ml, no more than 1 μg/ml or no more than0.5 μg/ml for binding to cells made to express at their surface saidallele of a human MICA polypeptide). Optionally, the antibodies bind toregions on the α1α2 domain that are located outside or partially outsidethe the NKG2D interface, but not completely within the NKG2D interface.

Binding to MICA alleles and related EC50 values can be assessed using,e.g., flow cytometry, according to the methods of Example 3 herein.

In another embodiment, the present invention results from the discoveryof antibodies that bind the α1 and/or α2 domain of MICA withoutsubstantially blocking the interaction of MICA with NKG2D (e.g. whereinMICA and NKG2D are each expressed at the surface of cells). Suchantibodies can optionally be characterized as not competing with hNKG2Din binding to MICA. Optionally the antibodies do not inhibit the abilityof MICA to induce NKG2D activity in a NKG2D-expressing cell. Suchantibodies can optionally be characterized as not decreasing or blockingthe ability of a NKG2D-expressing effector cell (e.g. a CD16-negativeeffector cell) to lyse a MICA-expressing target cell. The antibodieswill optionally not substantially block shedding of MICA from tumorcells.

In one embodiment, a non-blocking α1α2 domain antibody binds an epitopeon a MICA polypeptide of SEQ ID NO:1 comprising one or two residuesselected from the group consisting of K81 and D82, one or two residuesselected from the group consisting of Q83 and K84, one, two or threeresidues selected from the group consisting of H109, Y111 and L116,optionally residue D113, one, two or three residues selected from thegroup consisting of Q131, S132 and Q136, one, two or three residuesselected from the group consisting of S133, R134 and T137, and/or 1, 2,3 or 4 residues selected from the group consisting of M140, N141, R143and N144 (e.g. antibody 20C6 and 10A7).

In one embodiment, a non-blocking α1α2 domain antibody bind an epitopecomprising one or two residues selected from the group consisting of R6and N8, one or two residues selected from the group consisting of E97and H99, one, two or three residues selected from the group consistingof E100, D101 and N102, one, two or three residues selected from thegroup consisting of S103, T104 and R105, optionally residue E115, and/orone, two or three residues selected from the group consisting of L178,R179 and R180 (e.g. antibody 15F9).

In one embodiment, a non-blocking α1α2 domain antibody binds an epitopecomprising one or two residues selected from the group consisting of Q48and W49 of the MICA polypeptide of SEQ ID NO: 1, and/or 1, 2, 3 or 4residues selected from the group consisting of E51, D52, V53 and L54 ofthe MICA polypeptide of SEQ ID NO: 1 (e.g. antibody 6E4).

In another embodiment, the present invention results, inter alia, fromthe discovery of antibodies that bind the α3 domain of MICA, wherein theantibodies do not inhibit the interaction of MICA with NKG2D (e.g.wherein MICA and NKG2D are each expressed at the surface of cells).Optionally the antibodies do not substantially blocking shedding of MICAfrom tumor cells. Optionally, such an antibody can optionally becharacterized as not competing with hNKG2D in binding to MICA.

In one embodiment, a non-blocking α3 domain antibody binds an epitopecomprising one, two or three residues selected from the group consistingof S224, H225 and D226, one, two or three residues selected from thegroup consisting of T227, Q228 and 0229, and/or one or two residuesselected from the group consisting of W230 and D232 (e.g. antibody16A8).

In another embodiment, the present invention results from the discoveryof antibodies that bind the α1 and/or α2 domain of MICA and inhibit theinteraction of MICA with NKG2D (e.g. wherein MICA and NKG2D are eachexpressed at the surface of cells). Such an antibody can optionally becharacterized as competing with hNKG2D in binding to MICA. Theantibodies will optionally inhibit sMICA-induced downmodulation of NKG2Dexpression on the surface of an immune effector cell withoutsubstantially blocking shedding of MICA from tumor cells.

In one embodiment, a blocking α1α2 domain antibody binds an epitopecomprising one, two or three residues selected from the group consistingof E100, D101 and N102, one, two or three residues selected from thegroup consisting of S103, 1104 and R105, one or two residues selectedfrom the group consisting of N121 and E123, and/or one or two residuesselected from the group consisting of T124 and E126 (e.g. antibody 19E9,18E8 and 10F3).

In one embodiment, a blocking α1α2 antibody binds an epitope comprising1, 2 or 3 residues selected from the group consisting of N56, K57 andT58 of the MICA polypeptide of SEQ ID NO: 1, and/or one or two residuesselected from the group consisting of R61 and R64 of the MICApolypeptide of SEQ ID NO: 1 (e.g. antibody 9C10 and 12A10).

In another embodiment, the present invention results, inter alia, fromthe discovery of antibodies that bind the α3 domain of MICA, wherein theantibodies inhibit the interaction of MICA with NKG2D (e.g. wherein MICAand NKG2D are each expressed at the surface of cells). Optionally theantibodies do not substantially blocking shedding of MICA from tumorcells. Optionally, such an antibody can optionally be characterized ascompeting with hNKG2D in binding to MICA. The antibodies will optionallyinhibit sMICA-induced downmodulation of NKG2D expression on the surfaceof an immune effector cell. The antibodies may substantially blockshedding of MICA from tumor cells or may optionally not substantiallyblock shedding of MICA from tumor cells.

In one embodiment, a blocking α3 domain antibody binds an epitopecomprising one, two or three residues selected from the group consistingof T227, Q228 and 0229 (antibody 14B4).

Without wishing to be bound by theory, it is believed that despite thescientific literature which assumes a causal relationship between MICA(e.g., sMICA or membrane-bound MICA) and NKG2D downregulation andimpairment of effector cells, MICA does not itself in tumor settingsalways cause substantial impairment of effector cells. In particular,while sMICA can cause downregulation of NKG2D, the concentrations ofsMICA that occur in vivo may in many cases be too low to itself causesignificant NKG2D downregulation (see Example 9). Furthermore, in tumorsettings (e.g. established or advanced disease), the patient isgenerally in an immunosuppressed state via a number of non-MICAcomponents (e.g. TGF-beta) that have the potential, among other effects,to cause the downmodulation of NKG2D. Consequently, agents that block ordo not block NKG2D-MICA interactions and do not inhibit MICA sheddingare efficacious in treatment of cancers so long as they are capable ofinducing CDC and/or ADCC. Antibodies that do not block NKG2D-MICAinteractions be advantageous because MICA-expressing tumor cells havethe potential to remain recognizable by NKG2D on immunocompetenteffector cells that are present (e.g. as immunocompetence re-establishesin a patient during or subsequent to a treatment, for treatments havinga long duration, repeated administration or administered at high doses).Antibodies that block NKG2D-MICA interactions be advantageous in thatsuch antibodies may prevent the MICA-induced downregulation of NKG2D(see Example 9). Such blocking antibodies may be particularly useful forthe treatment of patients having high levels of soluble MICA, e.g. incirculation.

In one embodiment, the present invention provides a MICA bindingcompound, preferably an antibody that specifically binds to a MICApolypeptide (an anti-MICA antibody), without detectably reducing bindingbetween MICA and NKG2D (e.g., the interaction of surface MICA on tumorcells with surface NKG2D on effector cells), e.g., without substantiallyblocking the interaction of MICA and NKG2D. In one embodiment, thepresent invention provides a MICA binding compound (e.g. a MICA-bindingpolypeptide) that binds to a MICA polypeptide without substantiallyblocking shedding of MICA from tumor cells. In one embodiment, thepresent invention provides an MICA binding compound that binds to a MICApolypeptide without substantially blocking the interaction of MICA withNKG2D and without substantially blocking shedding of MICA from tumorcells.

In another embodiment, the present invention provides antibodies thatbind human MICA (particularly in the α1 and/or α2 domains) thatrecognize major MICA alleles MICA*001, MICA*004, MICA*008 and optionallyfurther MICA*007 and/or MICA*019. In one embodiment, the antibodiesoptionally further recognize MICA of a non-human primate specie (e.g.cynomolgus monkey). In one embodiment, the antibodies optionally furtherrecognize a MICB polypeptide comprising the amino acid sequence of SEQID NO 6. Optionally, in another embodiment, these antibodies further donot recognize MICB. Optionally the antibodies do not substantially blockshedding of MICA from tumor cells. Optionally the antibodies do notsubstantially block the interaction of MICA with NKG2D.

In one embodiment, the present invention provides an antibody thatspecifically binds to a glycosylated MICA polypeptide expressed by ahuman tumor cell.

In one embodiment, the present invention provides an antibody thatspecifically binds to a MICA polypeptide expressed by a non-humanprimate cell.

In one embodiment, the present invention provides an antibody thatspecifically binds to a MICA polypeptide (an anti-MICA antibody),wherein the antibody binds a polypeptide of SEQ ID NO 2 (MICA*004)and/or a polypeptide of SEQ ID NO 4 (MICA*008). In one embodiment, theantibody further binds a polypeptide of SEQ ID NO 1 (MICA*001). In oneembodiment, the present invention provides an antibody that specificallybinds to a MICA polypeptide, wherein the antibody binds a polypeptide ofSEQ ID NO 5 (MICA*019). In one embodiment, the antibody further binds apolypeptide of SEQ ID NO 3 (MICA*007). In one embodiment, the presentinvention provides an antibody that specifically binds to a MICApolypeptide, wherein the antibody binds a polypeptide of SEQ ID NO 2(MICA*004), a polypeptide of SEQ ID NO 4 (MICA*008) and a polypeptide ofSEQ ID NO 5 (MICA*019). In one embodiment, the present inventionprovides an antibody that specifically binds to a MICA polypeptide,wherein the antibody binds a polypeptide of SEQ ID NO 1 (MICA*001), apolypeptide of SEQ ID NO 2 (MICA*004), a polypeptide of SEQ ID NO 4(MICA*008), and a polypeptide of SEQ ID NO 5 (MICA*019), optionallyfurther wherein the antibody binds a polypeptide of SEQ ID NO 3(MICA*007). By binding to alleles MICA*001, -*004, and *008, (andadvantageously further *007 and *019) across both Group 1 and Group 2 ofMICA alleles, virtually the entire human population will be suitable fortreatment with such an anti-MICA agent of the invention. In anyembodiment, a polypeptide of SEQ ID NOS 1-5 may comprise an O-glycan(N-acetyllactosamine linked to serine or threonine). In any embodiment,a polypeptide of SEQ ID NOS 1-5 may comprise a core2 O-glycan (anO-glycan comprising an N-acetylglucosamine branch connected toN-acetylgalactosamine) and/or an N-linked glycan. In one embodiment, theantibody binds to a MICA polypeptide without substantially blocking theinteraction of MICA with NKG2D and/or without substantially blockingshedding of MICA from tumor cells. In one embodiment, the antibody bindsthe α1 and/or α2 domain of MICA. In one embodiment, the antibody bindsthe α3 domain of MICA.

Preferably the compound is an antibody, optionally a tetrameric antibodycomprising two Ig heavy chains and two Ig light chains. Preferably theantibody has binding affinity (K_(D)), optionally wherein bindingaffinity is bivalent, for a human MICA polypeptide at of less than 10⁻⁹M, preferably less than 10⁻¹⁹ M, or preferably less than 10⁻¹¹M.Preferably the antibody is a depleting antibody, optionally wherein theantibody induces ADCC and/or CDC toward a MICA-expressing tumor cell.

In a specific embodiment, the present invention provides an antibodythat mediates depletion of MICA-expressing tumor cells by an NK or Tcell (e.g., in vivo or in vitro) without substantially inhibitingNKG2D-mediated cytotoxicity of a hNKG2D-expressing NK or T cell.

In a specific embodiment, an antibody of the invention does not competewith hNKG2D in binding to MICA.

In a specific embodiment, when an antibody of the invention is bound toMICA on a MICA-expressing cell, the MICA-expressing cell does notsubstantially reduce the amount of cell-surface hNKG2D upon binding via,e.g., stimulating down-modulation and/or internalization of hNKG2D, hasa high affinity and slow off-rate, cross-reacts with cynomolgus and/orrhesus MICA, and is of a depleting isotype such as, e.g., human IgG1.

In one aspect, the invention provides an antibody that specificallybinds MICA, wherein the antibody has one or more (including anycombination thereof, or all of) of the following properties:

(a) has a Kd of less than 10⁻⁸ M, preferably less than 10⁻⁸ M, orpreferably less than 10⁻¹⁰M for binding to a MICA polypeptide;

(b) binds to at least one residue in the segment corresponding toresidues of a domain selected from the group consisting of 1-88, 89-181and 182-274 of the MICA polypeptide of SEQ ID NO: 1 and/or binds to anepitope (one or more amino acid residues on MICA) as described herein;

(c) binds to two, three, four or five of the MICA*001, *004, *007, *008,and *019 polypeptides, respectively comprising a sequence of SEQ ID NOS:1-5;

(d) does not substantially block shedding of MICA from tumor cells;

(e) does not substantially block the interaction of MICA with NKG2D(e.g., the interaction of surface MICA on tumor cells with surface NKG2Don effector cells);

(f) does not cause a substantial decrease in lysis of MICA-expressingcells by effector cells (e.g., NKG2D+CD16− NK cells);

(g) induces complement dependent cytoxicity (CDC) and/or antibodydependent cellular cytoxicity (ADCC) toward a cell that expresses MICAon its surface; and

(h) competes for binding to a MICA polypeptide with antibody 6E4, 20C6,16A8, 15F9 and 10A7.

In any of the embodiments herein, an antibody of the invention may becharacterized by any one or more features of (a)-(h), above.

In one embodiment, provided is a method of testing an anti-MICAantibody, said method comprising: (i) assessing whether the antibodyblocks shedding of MICA from MICA-expressing cells and/or (ii) assessingwhether the antibody blocks the interaction of MICA with NKG2D. Step (i)may optionally comprise bringing the antibody that binds a MICApolypeptide into contact with a cell expressing a MICA polypeptide. Step(ii) may optionally comprise bringing the antibody that binds a MICApolypeptide into contact with a MICA polypeptide (e.g. an isolatedpolypeptide or a polypeptide expressed on the surface of a cell), in thepresence of an NKG2D polypeptide (e.g. an isolated polypeptide or apolypeptide expressed on the surface of a cell).

In another embodiment, provided is a method of producing an antibodythat binds a MICA polypeptide in a mammalian subject, optionally for thetreatment of a cancer, said method comprising the steps of: a) providinga plurality of antibodies, optionally immunizing a non-human mammal withan immunogen comprising a MICA polypeptide; and b) performing aselection step to select an antibody from the plurality, the stepcomprises:

-   -   (i) testing whether an antibody binds to a human MICA        polypeptide, optionally one, two, three, four or all of the        polypeptides of SEQ ID NOS 1-5, and selecting the antibody if it        binds to a human MICA polypeptide(s); and/or    -   (ii) testing whether an antibody blocks shedding of MICA from        MICA-expressing cells, and selecting the antibody if it does not        block shedding; and/or    -   (iii) testing whether an antibody blocks the interaction of MICA        (e.g. surface MICA) with NKG2D, preferably testing wherein the        antibody causes a substantial decrease in lysis of        MICA-expressing cells by effector cells (e.g., NKG2D+CD16− NK        cells, and selecting the antibody if it does not block the        interaction of MICA (e.g. surface MICA) with NKG2D, preferably        Wherein the antibody does not cause a substantial decrease in        lysis of MICA-expressing cells.

In one aspect, the invention results, inter alia, from the discovery ofblocking anti-MICA antibodies having high affinity across the majorhuman MICA alleles from the two main groups of MICA alleles (as well ason non-human primate MICA and MICB). In one embodiment, MICA groups arecharacterized by the presence of a methionine (M) or valine (V) residueat position 129 of the MICA polypeptide, wherein M is associated with aMICA form that binds strongly to NKG2D and V is associated with lowerbinding to NKG2D. In one embodiment, the invention provides blockingantibodies that cross-react with a MICA allele having a methionine atposition 129 and a MICA allele having a valine at position 129. In oneaspect the invention provides a monoclonal antibody that specificallybinds to a human MICA polypeptide having a methionine at position 129and a human MICA polypeptide having a valine at position 129, whereinsaid antibody inhibits MICA-mediated hNKG2D activity. In one embodimentthe antibody further binds antibody binds to a MICB polypeptide having avaline at position 152 (e.g., to a MICB polypeptide of SEQ ID NO: 1).Preferably, said antibody inhibits MICB-mediated hNKG2D activity. MICBpolypeptides having a valine at position 152 are reported to show strongbinding to soluble NKG2D. Antibodies that bind MICB polypeptides havingvaline at position 152 and MICA polypeptides having methionine atposition 129 may be advantageous in individuals who have greatersusceptibility or severe disease arising from alleles with strongbinding to NKG2D.

The blocking anti-MICA antibodies of the invention also cross-react(bind) one or more high prevalence alleles from each of two major MICAgroups that are determined to represent the main families of MICA: group1 alleles that bind NKG2D strongly (including MICA*001, *002, *007,*012, *017 and *018) and group 2 that bind NKG2D weakly (MICA*004, *006,*008, *009 and *019). By binding to an epitope present on the subsetMICA *001, *004, *007, *008 and *019, the antibodies cover the allelesof both groups that are present in almost all individuals. Group 1alleles of MICA generally have M at residue 129 while Group 2 alleleshave V at residue 129.

In another aspect, the present invention results, inter alia, from thediscovery of antibodies against MICA (as well as on non-human primateMICA and MICB) against certain epitopes are significantly more efficientin NKG2D blockade that either other anti-MICA antibodies or anti-NKG2Dantibodies, and are also more effective in blocking NKG2D-mediatedcytotoxicity than what would be expected from their ability to bindMICA. In particular, anti-MICA antibodies with affinities in thepicomolar range were 4-log better in inhibition than anti-MICAantibodies having a 2-log lesser affinity (K_(D)).

Blocking MICA antibodies of the invention may be characterized by theability to prevent any competition with, displacement by, or residualbinding of, NKG2D. Other high affinity NKG2D or MICA antibodies mayleave open some displacement by their ligand (e.g., MICA/ULPBs/RAET1 orNKG2D, respectively). Also, as observed from crystal structures of theNKG2D-MICA interaction, NKG2D acts as a homo-dimer and has twosymmetrical surfaces (one on each NKG2D chain) that interact with thetop of the MICA α1α2 platform domain. The two NKG2D chains bothcontribute to the interaction by binding to distinctly differencessurfaces of the asymmetric MICA platform domain (see e.g., Li et al.(2001) Nature Immunol. 2(5):443-450). The antibodies of the inventionmay thus optionally block the MICA-NKG2D interaction fully, e.g., byblocking (e.g. interfering, competing with) the binding of both NKG2Dmonomers in an NKG2D homodimer to a MICA polypeptide. Other MICAantibodies (or NKG2D antibodies) may block completely only one of thetwo NKG2D binding sites.

Treatment with the blocking anti-MICA antibodies of the invention, inaddition to their use to deplete MICA-expressing cells when used asdepleting antibody (e.g. IgG1 or IgG3 isotype), provides a means totarget MICA (and MICB) in inflammatory conditions believed to be drivenby MICA and/or B-mediated activation of NKG2D, without reducing causingunwanted broader immune system inhibition by reducing the number ofNKG2D receptors available for binding to other ligands (e.g. ULBPs) andsubsequent activation. The possibility of such fully blocking anti-MICAantibodies also open the possibility of local delivery of anti-MICAantibodies to sites of inflammation (e.g. into joints or other sites ofinflammation in arthritis patients) without causing a generalizedimmunosuppressive effect caused by administration of NKG2D antibodies.

In one embodiment, the present invention antibodies that are effectivein vitro and in vivo in inhibiting effector cell lysis (e.g. NK cellsand/or T cells) of MICA-expressing cells and substantially block theinteraction of MICA with NKG2D.

In one embodiment, particularly when used in the treatment ofinflammatory or autoimmune disorders, the antibody of the invention is anon-depleting antibody that substantially reduces or inhibits NKG2Dactivation, NKG2D-signalling, activation of NKG2D-expressing NK or Tcells, or lysis of MICA-expressing cells by effector cells (e.g.,NKG2D+CD16− NK cells).

In one embodiment, the present invention provides a MICA bindingcompound, preferably an antibody that specifically binds to a MICApolypeptide (an anti-MICA antibody) and reduces (e.g. substantiallyeliminates) binding between MICA and hNKG2D (e.g., the interaction ofsurface MICA on cells with surface NKG2D on effector cells).

In one embodiment, an anti-MICA antibody or binding compound furtherspecifically binds to a MICB polypeptide and reduces (e.g. substantiallyeliminates) binding between MICB and hNKG2D.

In one embodiment, the invention provides an antibody that competes withhNKG2D in binding to MICA (and optionally MICB) and prevents hNKG2D frombinding to MICA (and optionally MICB).

In one embodiment, the invention provides an antibody that inhibits orblocks the interaction of a MICA (and optionally MICB) polypeptide withboth hNKG2D chains of an hNKG2D homodimer. Optionally, the antibodyblocks the interaction of both an α1 domain of a MICA polypeptide (andoptionally MICB polypeptide) and an α1 domain of a MICA polypeptide (andoptionally MICB polypeptide), with an hNKG2D homodimer (e.g, theantibody inhibits the interaction of a MICA α1α2 platform with bothhNKG2D chains of an hNKG2D homodimer).

In one embodiment, an antibody of the invention does not substantiallybind to a HCMV, UL18, ULBP1, ULBP 2, ULBP 3, ULBP 4, ULBP 5 or ULBP 6polypeptide (see Champsaur et al (2010) Immunol. Rev. 235: 267-285).

In one embodiment, the present invention provides an antibody thatspecifically binds to a common determinant on a human MICA polypeptide(e.g. a naturally occuring MICA allele) having a methionine at position129 and a human MICA polypeptide (e.g. a naturally occuring MICA allele)having a valine at position 129, optionally further specifically bindsto a common determinant on a human MICB polypeptide (e.g. a naturallyoccuring MICB allele) having a valine at position 152 wherein theantibody reduces (e.g. substantially eliminates) binding between MICAand NKG2D (and optionally between MICB and NKG2D).

In a further embodiment, the present invention results, inter alia, fromthe discovery of antibodies that bind human MICA, preferably in the α1α2platform domain (i.e. the α1 and/or α2 domain), and that recognize majorMICA alleles MICA*001, MICA*004, MICA*008 and optionally furtherMICA*007 and/or MICA*019, and optionally further recognize MICA of anon-human primate specie (e.g. cynomolgus monkey), and optionallyfurther recognize MICB. In one embodiment, the antibodies furtherrecognize a MICB polypeptide comprising an amino acid sequence of SEQ IDNO: 6. Optionally the antibodies furthermore do not substantiallyinhibit shedding of MICA from tumor cells.

In one embodiment, the present invention provides an antibody thatspecifically binds to a MICA polypeptide (an anti-MICA antibody),wherein the antibody reduces (e.g. substantially eliminates) bindingbetween MICA and NKG2D and wherein the antibody binds a polypeptide ofSEQ ID NO: 2 (MICA*004) and/or a polypeptide of SEQ ID NO: 4 (MICA*008).In one embodiment, the antibody further binds a polypeptide of SEQ IDNO: 1 (MICA*001). In one embodiment, the present invention provides anantibody that specifically binds to a MICA polypeptide, wherein theantibody binds a polypeptide of SEQ ID NO: 5 (MICA*019). In oneembodiment, the antibody further binds a polypeptide of SEQ ID NO: 3(MICA*007). In one embodiment, the present invention provides anantibody that specifically binds to a MICA polypeptide, wherein theantibody binds a polypeptide of SEQ ID NO: 2 (MICA*004), a polypeptideof SEQ ID NO: 4 (MICA*008) and a polypeptide of SEQ ID NO: 5 (MICA*019).In one embodiment, the present invention provides an antibody thatspecifically binds to a MICA polypeptide, wherein the antibody binds apolypeptide of SEQ ID NO: 1 (MICA*001), a polypeptide of SEQ ID NO: 2(MICA*004), a polypeptide of SEQ ID NO: 4 (MICA*008), and a polypeptideof SEQ ID NO: 5 (MICA*019), optionally further wherein the antibodybinds a polypeptide of SEQ ID NO: 3 (MICA*007). By binding to allelesMICA*001, -*004, and *008, (and advantageously further *007 and *019)across both Group 1 and Group 2 of MICA alleles, virtually the entirehuman population will be suitable for treatment with such an anti-MICAagent of the invention. In one embodiment, the antibody binds the α1and/or α2 domain of MICA.

Preferably the compound is an antibody, optionally a tetrameric antibodycomprising two Ig heavy chains and two Ig light chains.

Preferably the antibody has binding affinity (K_(D)), optionally whereinbinding affinity is monovalent or bivalent, for a human MICA and/or MICBpolypeptide (e.g. any one or more or all MICA and/or MICB allelesreferred to herein) of less than 10⁻⁹ M, preferably less than 10⁻¹⁰ M,preferably less than 10⁻¹¹ M, preferably less than 10⁻¹² M, orpreferably less than 10⁻¹³ M. Preferably the antibody is a tetramericantibody comprising two Ig heavy chains and two Ig light chains and theK_(D) is bivalent. Optionally, the antibody has an EC50 for binding tocells made to express a particular MICA and/or MICB polypeptide, forexample any one or more or all MICA and/or MICB alleles referred toherein) of no more than 10, 5, or 1 μg/ml. Examples of suitable cellsare C1R-MICA cells.

In one embodiment, particularly when the antibody is for use in treatingor preventing inflammatory or autoimmune disease, the compound is anon-depleting antibody (an antibody that does not deplete cells to whichit binds). Preferably the antibody is a chimeric, humanized or humanantibody. Preferably the antibody does not comprise a constant regioncapable of being bound by an Fcγ3A receptor (CD16), e.g. an antibody ofthe human IgG4 subtype or an antibody fragment lacking an Fc domain.Preferably the antibody comprises a heavy chain constant region of humanIgG4 isotype.

In one embodiment, the antibody specifically binds to a MICA polypeptideexpressed by a non-human primate cell. In a specific embodiment, anantibody of the invention has a high affinity and slow off-rate forbinding to human MICA, and cross-reacts with cynomolgus (Macacafascicularis) and/or rhesus (Macaca mulatta) MICA.

In one aspect, the invention provides an antibody that specificallybinds MICA, wherein the antibody has one or more (including anycombination thereof, or all of) of the following properties:

(a) has a K_(D) of less than 10⁻⁹ M, preferably less than 10⁻¹⁰ M,preferably less than 10⁻¹¹ M, preferably less than 10¹² M, or preferablyless than 10⁻¹³M for binding to a MICA polypeptide, preferably whereinthe antibody has an affinity of said K_(D) for each MICA polypeptidealleles MICA*001, *004, and *008, optionally further *007 and/or *019;

(b) binds to at least one residue in the segment corresponding toresidues of a domain selected from the group consisting of 1-88 or89-181 of the MICA polypeptide of SEQ ID NO: 1, and/or binds to anepitope (residues on MICA) as described herein;

(c) binds to a human MICA polypeptide (e.g. a naturally occuring MICAallele) having a methionine at position 129 and a human MICA polypeptide(e.g. a naturally occuring MICA allele) having a valine at position 129.wherein the antibody reduces (e.g. substantially eliminates) binding.between MICA and NKG2D; and/or binds to a human MICB polypeptide (e.g. anaturally occuring MICA allele) having a valine at position 152;

(d) binds to two, three, four or five of the MICA*001, *004, *007, *008,and *019 polypeptides, respectively comprising a sequence of SEQ ID NOS:1-5;

(e) does not substantially block shedding of MICA from tumor cells;

(f) inhibit and/or substantially block the interaction of MICA withNKG2D (e.g., the interaction of surface MICA on tumor cells with surfaceNKG2D on effector cells), optionally wherein the antibodies block theinteraction of MICA with both NKG2D chains within a NKG2D homodimer.

(g) are capable of reducing or inhibiting MICA-mediated NKG2Dactivation, NKG2D-signalling, activation of NKG2D-expressing NK or Tcells, or lysis of MICA-expressing cells by effector cells (e.g.,NKG2D+CD16− NK cells);

(h) does or does not (e.g. depending on whether the mAb is found byCD16, depending on the nature of the Fc region of the antibody)substantially induces complement dependent cytotoxicity (CDC) and/orantibody dependent cellular cytotoxicity (ADCC) toward a cell thatexpresses MICA on its surface;

(i) are capable of reducing or inhibiting sMICA-mediated downmodulationof NKG2D expression on the surface of NKG2D-expression cells (e.g. Tcells or NK cells);

and

(j) competes for binding to a MICA polypeptide with antibody 9C10, 19E9,12A10, 18E8 or 10F3.

In any of the embodiments herein, an antibody of the invention may becharacterized by any one or more features of (a)-(j), above.

In one embodiment, provided is a method of testing an anti-MICAantibody, said method comprising: (i) assessing whether the antibodybinds with high affinity to both a MICA polypeptide having a methionineat residue 129 and a MICA polypeptide having a valine at residue 129(and optionally further a MICB polypeptide having a methionine atresidue 152), and (ii) assessing whether the antibody blocks theinteraction of MICA (and optionally MICB) with NKG2D. In one embodiment,provided is a method of testing an anti-MICA antibody, said methodcomprising: (i) assessing whether the antibody binds with high affinityto two, three, four or five of the MICA*001, *004, *007, *008, and *019polypeptides, respectively comprising a sequence of SEQ ID NOS: 1-5 and(ii) assessing whether the antibody blocks the interaction of MICA withNKG2D. Step (i) may optionally comprise bringing the antibody that bindsa MICA polypeptide into contact with a cell expressing a MICApolypeptide. Step (ii) may optionally comprise bringing the antibodythat binds a MICA polypeptide into contact with a MICA polypeptide (e.g.an isolated polypeptide or a polypeptide expressed on the surface of acell), in the presence of an NKG2D polypeptide (e.g. an isolatedpolypeptide or a polypeptide expressed on the surface of a cell). Anantibody that binds said MICA polypeptides in step (i) and blocks theinteraction of MICA with NKG2D in step (ii) can be identified and/orselected as a candidate treatment for an inflammatory or autoimmunedisorder.

In another embodiment, provided is a method of producing an antibodythat binds a MICA polypeptide in a mammalian subject, optionally for thetreatment of a cancer, said method comprising the steps of: a) providinga plurality of antibodies, optionally immunizing a non-human mammal withan immunogen comprising a MICA polypeptide or producing a phage displaylibrary of antibodies; and b) performing a selection step to select anantibody from said plurality, the step comprising:

(i) testing whether an antibody binds with high affinity to a human MICApolypeptide, optionally one, two, three or all of the MICA*001, *004,*007, *008, and *019 alleles, e.g., polypeptides respectively comprisinga sequence of SEQ ID NOS: 1-5, and selecting an antibody if it bindswith high affinity to said MICA polypeptide; and/or

(ii) testing whether an antibody blocks the interaction of MICA (e.g.surface MICA, any one, two, three or all of the MICA*001, *004, *007,*008, and *019 alleles) with hNKG2D, and/or reduces or inhibits NKG2Dactivation, NKG2D-signalling, activation of NKG2D-expressing NK or Tcells, or lysis of MICA-expressing cells by effector cells (e.g., NKG2D+T cells, NKG2D+CD16+NK cells, NKG2D+CD16− NK cells), and selecting anantibody if it blocks the interaction of MICA with hNKG2D; and/or.

(iii) testing whether an antibody reduces or inhibits NKG2Ddownmodulation (decrease of cell surface expression) in aNKG2D-expressing cell when said NKG2D-expressing cell is brought intocontact with soluble MICA polypeptide in the presence of the antibody,and selecting an antibody if it antibody reduces or inhibits NKG2Ddownmodulation.

The method may optionally comprise a selection step (iv) comprisingtesting whether an antibody blocks shedding of MICA from MICA-expressingcells, and selecting the antibody if it does not block shedding.

In one embodiment, an antibody of the invention binds an epitopecomprising 1, 2, 3, 4, 5, 6, 7 or more residues of a human MICApolypeptide selected from the group consisting of R6, N8, Q48, W49, E51,D52, V53, L54, N56, K57, T58, R61, R64, K81, D82, Q83, K84, E97, H99,E100, D101, N102, S103, T104, R105, H109, Y111, D113, E115, L116, N121,E123, T124, E126, Q131, S132, S133, R134, Q136, T137, M140, N141, R143,N144, L178, R179, R180, S224, H225, D226, T227, Q228, Q229, W230 andD232 (with reference to a MICA of any of SEQ ID NOS 1-5).

In one embodiment, the present invention provides a MICA bindingcompound, preferably an anti-MICA antibody, which binds at least partly,or optionally completely, within the α1 and/or α2 domain of MICApolypeptides. The α1 and α2 domains are located within amino acidresidues 1 to 88 (optionally 1-85) and 89 to 181 (optionally 86-181),respectively, with reference to the MICA polypeptide of SEQ ID NO 1.Optionally, in any of the embodiments herein, the antibody binds to anamino acid residue within the α1 domain of a MICA polypeptide (residuesamino acid residues 1 to 88 (optionally 1-85) of SEQ ID NO 1).Optionally, in any of the embodiments herein, the antibody binds to anamino acid residue within the α2 domain of a MICA polypeptide (residuesamino acid residues 89 to 181 (optionally 86-181) of SEQ ID NO 1).Optionally, in any of the embodiments herein, the antibody binds toamino acid residues within the α1 and α2 domain of a MICA polypeptide.

In one embodiment, an antibody binds an epitope comprising one or tworesidues selected from the group consisting of Q48 and W49, and/or 1, 2,3 or 4 residues selected from the group consisting of E51, D52, V53 andL54 (antibody 6E4).

In one embodiment, an antibody binds an epitope comprising 1, 2 or 3residues selected from the group consisting of N56, K57 and T58, and/orone or two residues selected from the group consisting of R61 and R64(antibody 9C10 and 12A10).

In one embodiment, an antibody binds an epitope comprising one or tworesidues selected from the group consisting of K81 and D82, one or tworesidues selected from the group consisting of Q83 and K84, one, two orthree residues selected from the group consisting of H109, Y111 andL116, optionally residue D113, one, two or three residues selected fromthe group consisting of S133, R134 and T137, and/or 1, 2, 3 or 4residues selected from the group consisting of M140, N141, R143 and N144(antibody 20C6).

In one embodiment, an antibody binds an epitope comprising one or tworesidues selected from the group consisting of K81 and D82, one or tworesidues selected from the group consisting of Q83 and K84, one, two orthree residues selected from the group consisting of H109, Y111 andL116, optionally residue D113, one, two or three residues selected fromthe group consisting of Q131, S132 and Q136, and/or 1, 2, 3 or 4residues selected from the group consisting of M140, N141, R143 and N144(antibody 10A7).

In one embodiment, an antibody binds an epitope comprising one, two orthree residues selected from the group consisting of E100, D101 andN102, one, two or three residues selected from the group consisting ofS103, T104 and R105, one or two residues selected from the groupconsisting of N121 and E123, and/or one or two residues selected fromthe group consisting of T124 and E126 (antibody 19E9 and 18E8).

In one embodiment, the antibodies bind an epitope comprising one or tworesidues selected from the group consisting of R6 and N8, one or tworesidues selected from the group consisting of E97 and H99, one, two orthree residues selected from the group consisting of E100, D101 andN102, one, two or three residues selected from the group consisting ofS103, T104 and R105, optionally residue E115, and/or one, two or threeresidues selected from the group consisting of L178, R179 and R180(antibody 15F9).

In one embodiment, the present invention provides a MICA bindingcompound, preferably an anti-MICA antibody, that binds at least partlywithin the α3 domain of MICA polypeptides. The α3 domain is locatedwithin amino acid residues 182 to 274, respectively, with reference tothe MICA polypeptide of SEQ ID NO 1. In one embodiment, an antibodybinds an epitope comprising one, two or three residues selected from thegroup consisting of T227, Q228 and Q229 (antibody 14B4 and 16A8). In oneembodiment, an antibody binds an epitope comprising one, two or threeresidues selected from the group consisting of S224, H225 and D226, one,two or three residues selected from the group consisting of T227, Q228and Q229, and/or one or two residues selected from the group consistingof W230 and D232 (antibody 16A8).

Optionally, binding of the antibody to a MICA polypeptide having amutation at any of the foregoing residues within the α1 and/or α2 domainis substantially reduced, in comparison to binding to a wild-type MICApolypeptide of SEQ ID NO: 1.

The present invention provides that the use of anti-MICA antibodies canbe useful for the treatment of cancers, inflammatory and autoimmunedisorders, e.g. in human subjects.

In one aspect, an antibody an antibody that does not inhibit theinteraction between NKG2D and MICA, or an antibody that inhibits theinteraction between NKG2D and MICA, will be a depleting antibody. Suchantibodies are particularly useful in the treatment of cancers but mayalso be used in inflammation and autoimmune disorders. Antibodies can beused with or without coupling to a toxic or other agent, depending onthe desired effect or use made of the antibodies. In one embodiment, theanti-MICA antibody is a “naked antibody” and is not coupled to a toxicagent, optionally the naked antibody comprises an Fc region modified toincrease binding to an Fcγ receptor, e.g., CD16. In one embodiment, anaked or coupled antibody comprises a heavy chain comprising a human Fcregion (e.g. IgG1) that binds Fcγ receptors (e.g. CD16). Optionally suchantibody induces complement dependent cytoxicity (CDC) and/or antibodydependent cellular cytoxicity (ADCC) toward a cell that expresses MICAon its surface.

Optionally, in any embodiment, the antibody (e.g. IgG1, antibodyfragment, etc.) further comprises a toxic agent (e.g. a chemotherapeuticagent) that is toxic to a cell.

In one aspect, an antibody an antibody that inhibits MICA-induced NKG2Dactivity in an effector cell, will be a non-depleting antibody (anantibody that does not deplete cells to which it binds). In one aspectthe antibody is a chimeric, humanized or human antibody. In one aspectthe antibody does not comprise a constant region capable of being boundby an Fcγ3A receptor (CD16), e.g. an antibody of the human IgG4 subtypeor an antibody fragment lacking an Fc domain. In one embodiment, theantibody comprises an IgG4 heavy chain comprising a serine to prolinemutation in residue 228 according to the EU-index. Preferably theantibody comprises a heavy chain constant region of human IgG4 isotype.Such antibodies are particularly useful in the treatment of inflammationand autoimmune disorders.

The present disclosure further provides antibodies, antibody fragments,and derivatives that specifically bind human MICA. The inventionprovides such antibody compositions, as well their use in any of themethods of the invention of treating, preventing and diagnosing cancer,inflammatory disorders and autoimmune disorders.

In one embodiment, the antibodies have binding affinity (K_(D)) for ahuman MICA polypeptide (e.g., a polypeptide of one, two, three or all ofthe MICA*001, *004, *007, *008, and *019 alleles of SEQ ID NOS 1-5,preferably to each of MICA*001, *004 and *008) of less than 10⁻⁸ M,preferably less than 10⁻⁹ M, or preferably less than 10⁻¹⁰M.

In one aspect of any of the embodiments of the invention, the antibodymay have a heavy and/or light chain having one, two or three CDRs of therespective heavy and/or light chain of an antibody selected from thegroup consisting of antibody 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7,18E8, 10F3, 15F9 and 14B4.

In one aspect of any of the embodiments of the invention, the antibodycompetes for binding to a MICA polypeptide with any one or anycombination of monoclonal antibodies 6E4, 20C6, 16A8, 9C10, 19E9, 12A10,10A7, 18E8, 10F3, 15F9 and 14B4. In one embodiment, an antibody of theinvention competes for binding to a MICA polypeptide, with an antibodyselected from the group consisting of:

-   -   (a) an antibody having respectively a VH and VL region of SEQ ID        NOS: 7 and 8 (6E4);    -   (b) (a) an antibody having respectively a VH and VL region of        SEQ ID NOS: 20 and 21 (20C6);    -   (c) an antibody having respectively a VH and VL region of SEQ ID        NOS:33 and 34 (16A8);    -   (d) an antibody having respectively a VH and VL region of SEQ ID        NOS: 46 and 47 (19E9);    -   (e) an antibody having respectively a VH and VL region of SEQ ID        NOS: 57 and 58 (9C10);    -   (f) an antibody having respectively a VH and VL region of SEQ ID        NOS: 68 and 69 (12A10);    -   (g) an antibody having respectively a VH and VL region of SEQ ID        NOS: 79 and 80 (10A7);    -   (h) an antibody having respectively a VH and VL region of SEQ ID        NOS: 90 and 91 (18E8);    -   (i) an antibody having respectively a VH and VL region of SEQ ID        NOS: 101 and 102 (10F3);    -   (j) an antibody having respectively a VH and VL region of SEQ ID        NOS: 112 and 113 (15F9); and    -   (k) an antibody having respectively a VH and VL region of SEQ ID        NOS: 123 and 124 (14E34).

In one embodiment, the antibody is human-suitable. In one embodiment theantibody is chimeric, e.g. contains a non-murine, optionally a human,constant region. In one embodiment, the antibody is human or humanized.In one aspect of any of the embodiments of the invention, the isotype ofthe antibody is a human IgG, optionally human IgG1, IgG2, IgG3 or IgG4.In one embodiment the antibody comprises a human Fc domain or is of anisotype that is bound by FcγR (e.g. FcγRIIIA), e.g. an antibody of IgG1or IgG3 isotype.

In one aspect of any of the embodiments of the invention, the antibodyis an antibody fragment selected from Fab, Fab′, Fab′-SH, F(ab′)2, Fv,diabodies, single-chain antibody fragment, or a multispecific antibodycomprising multiple different antibody fragments. Optionally antibodiesof the invention are furthermore tetrameric (two heavy and two lightchains) and are thus bivalent (e.g. IgG antibodies).

In certain embodiments, the antibodies of the invention further comprisea toxic agent. In one embodiment, the antibodies comprising a toxicagent are able to directly cause the death of cells expressing MICA. Inone embodiment, the antibodies are capable of directly inducing (e.g. inthe absence of immune effector cells) at least 20%, 30%, 40% or 50% celldeath, e.g. in an in vitro assay, of MICA-expressing cells.

In one aspect, the invention provides methods of treatment using theanti-MICA antibodies of the invention. The antibodies can be used asprophylactic or therapeutic treatment; in any of the embodiments herein,a therapeutically effective amount of the antibody can be interchangedwith a prophylactically effective amount of an antibody. In one aspect,the invention provides a method of treating a patient with a cancer, anautoimmune disorder or an inflammatory disorder, the method comprisingadministering to the patient a pharmaceutically effective amount of anantigen-binding compound according to the invention that specificallybinds to a MICA polypeptide.

The methods of treatment of the invention and the anti-MICA antibodyaccording to the invention can be used to a treat an individual incombination with a second therapeutic agent, including an anti-canceragent when used to treat cancer (e.g. chemotherapeutic drugs, tumorvaccines, antibodies that bind to tumor-specific antigens on tumorcells, antibodies that induce ADCC toward tumors cells, antibodies thatpotentiate immune responses, etc.), or an agent useful for the treatmentof autoimmunity or inflammation. In one embodiment, the secondtherapeutic agent is an agent (e.g. an antibody) that binds to andactivates an activating receptor or that binds to and blocks aninhibitory receptor on an effector cell (e.g. an NK cell, a T cell). Inone embodiment, the second therapeutic agent is an agent (e.g. achemotherapeutic agent) that upregulates the expression of an NKG2Dligand on tumor cells. For example, histone deacetylase inhibitors canbe used. For example, valproate and hydralazine augment MICA/Bexpression and decrease shedding. (Chavez-Blanco 2011 Int J Oncol 39(6):1491-1499).

The presence of increased levels of sMIC in circulation has beenassociated with poor prognosis and/or MIC expressing tumors. The presentinvention further concerns a method for selecting subjects having acancer that responds to a treatment using an anti-MICA agent of theinvention (e.g. an antibody that binds to a MICA polypeptide), themethod comprising determining whether tumor cells in said subject shed aMICA polypeptide (e.g. as assessed by levels of sMIC in circulation),the presence of shedding of MICA polypeptide from tumor cells beingindicative of a responder subject.

The present invention further concerns a method for selecting subjectshaving a disorder (e.g., a cancer, an autoimmune disorder, aninflammatory disorder) that responds to a treatment using an anti-MICAagent of the invention (e.g. an antibody that binds to a MICApolypeptide), the method comprising determining whether cells (e.g.tumor cells, pro-inflammatory cells, etc.) in said subject express aMICA polypeptide, the expression of a MICA polypeptide being indicativeof a responder subject. In one embodiment, the method comprisesdetermining whether cells (e.g. tumor cells, pro-inflammatory cells,etc.) in said subject express a MICA polypeptide selected from the groupconsisting of SEQ ID NOS 1-5. In one embodiment, the method comprisesdetermining whether cells in said subject express a MICA polypeptideselected from the group consisting of MICA*001, *004, *007, *008, and*019 polypeptides, respectively comprising a sequence of SEQ ID NOS:1-5, wherein the expression of a MICA polypeptide is indicative of aresponder subject. In one embodiment, the step of determining whethercells in said subject express a MICA polypeptide comprising bringing abiological sample from the subject (e.g. by performing a biopsy and/orobtaining a sample of cancer cells, a blood or any tissue sample, etc.)into contact with an anti-MICA antibody of the invention (e.g. anantibody that bind a one, two, three, four or all of the MICA*001, *004,*007, *008, and *019 polypeptides). In one embodiment, the methodcomprises determining whether said subject comprises shed MICA (e.g.detecting sMICA in circulation or detecting the presence of MICA on thesurface of exosomes (*008 allele, for example).

Optionally, in any of the methods, the method further comprisesadministering to a responder subject an antibody (e.g. an anti-MICAantibody of the invention) that binds to a MICA polypeptide.

In a preferred embodiment, the expression of a MICA polypeptide in adisease-related cell is determined using a MICA-specific ligand.Preferably, the ligand is an antibody, or a fragment or derivativethereof.

In another aspect, the invention comprises a method (e.g., a method ofconducting a diagnostic assay, a responder assay, etc.), comprisingassessing whether a patient has disease-related cells (e.g., tumorcells) expressing a MICA polypeptide, e.g. a MICA polypeptide (one ormore MICA alleles) bound by an antibody of the invention. Said methodmay comprise, for example, obtaining a biological sample from a patientcomprising disease-related cells, bringing said disease-related cellsinto contact with such antibody and assessing whether the antibody bindsto disease-related cells. A finding that MICA is expressed bydisease-related cells indicates that the patient has a conditioncharacterized by MICA-expressing cells and/or is suitable for treatmentwith an anti-MICA antibody of the invention. The patient can further betreated with a treatment suitable for the particular diseasecharacterized by MICA-expressing cells. Optionally the patient istreated with the anti-MICA antibody. In one embodiment, the method isused for selecting subjects having a cancer, and the disease-relatedcells are cancer cells.

The present invention also provides a method of treating a patient, themethod comprising:

a) determining whether the patient has pathogenic MICA-expressing cells,and b) if the patient is determined to patient have pathogenicMICA-expressing cells, administering an antigen-binding compound (e.g.,antibody) of the invention.

The present invention also provides a method of treating a patient, themethod comprising:

-   -   a) determining the level of shed MICA in the patient, and    -   b) if the patient is determined to patient have elevated levels        of shed MICA, administering an antigen-binding compound (e.g.,        antibody) of the invention that inhibits the ability of sMICA to        cause downmodulation of NKG2D on an immune effector cell.

These and additional advantageous aspects and features of the inventionmay be further described elsewhere herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show binding of antibodies obtained from the first,second and third immunization series to MICA-expressing CR1 transfectantcells C1R-MICA*001, C1R-MICA*004, C1R-MICA*007 and C1R-MICA*008, asanalysed by flow cytometry.

FIGS. 2A-2G show views of the MICA polypeptide, including in darkshading the amino acid residues mutated which resulted (in differentcombinations) in loss of binding by antibodies. The NKG2D binding siteis shown at the top of the MICA polypeptides in medium shading (and alsoin ribbon diagrams bound to MICA)

FIG. 3 shows superimposed sensorgrams showing the injections ontoNKG2D-Fc chip of MICA*01-BirA alone or pre-incubated with isotypecontrol or chimeric anti-MICA antibodies. Sensorgrams were normalized inthe Y axis and aligned in the X axis at the end of injection.Sensorgrams are representative of two independent experiments.

FIG. 4 shows the results of a functional assay for MICA-NKG2A blockade,showing the ability of anti-MICA antibodies to reduce or inhibit theNKG2D+CD16− NK92 cell-mediated killing of MICA*019-transfected BaF/3 asdetermined by measuring target cell release of ⁵¹Cr.

FIGS. 5A, 5B and 5C show superimposed sensorgrams showing the injectionsonto ULBP-1-Fc (Fc1), MICB-Fc (Fc2), ULBP-2-Fc (Fc3) and ULBP-3-Fc (Fc4)of Anti-MICA antibodies. Sensorgrams were aligned in the X axis at thestart of injection.

FIG. 6 shows binding of anti-MICA antibodies to Macaca fascicularisMICA.

FIG. 7 shows inhibition of the MICA shedding mediated by the anti-α3domain BAM03 but not by 16A8, in an assay for capacity to block MICAshedding as assessed by measuring soluble MICA concentration in thesupernatant after overnight incubation of MICA-expressing cells withanti-MICA antibodies.

FIG. 8 shows that NKG2D is downmodulated by 30 to 40% of its initiallevel in presence of increasing doses of recombinant bivalentMICA*019*Fc protein (R&D systems), and that anti-MICA antibodies,blocking NKG2D-MICA interaction in the cytotoxic assay of Example 5B(Table E), are blocking the interaction of NKG2D expressed on NK cellswith MICA*019-Fc, hence reversing the NKG2D downmodulation induced bythe MICA*019-Fc protein.

FIG. 9 shows viability of indicated Raji-MICA*01 cells, in the presenceof human complement. The results show that anti-MICA (isotype matched)cause an increase the number of dead cells and are capable of inducingCDC in an epitope dependent fashion.

FIG. 10A shows that anti-MICA each induced specific lysis ofC1R-MICA*008 cells by human KHYG-1 hCD16V NK cells line compared tonegative controls (Human IgG1 isotype control antibody), thereby showingthat these antibodies induce ADCC toward MICA*008-expressing targetcells. FIG. 10B shows level of antibody binding to the cell.

FIG. 11 shows results of testing of in a mouse long-term RAJI-MICA*01tumor model. Survival of Nod SCID mice engrafted Raji-MICA01 High 15M IVtreated with chimeric anti-MICA or IC (3004 IP 2×/week for 3 weeks) orPBS. Anti-MICA antibodies of the invention increased survival.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The antibodies of the invention are able to directly and specificallytarget MICA-expressing cells, notably tumor cells and cells involved ininflammatory or autoimmune processes. The invention provides a number ofantibodies having such properties which bind both Group 1 and Group 2alleles and moreover across the principal human MICA alleles withinthese groups. Further provided are different antibodies useful forparticular approaches. Some antibodies block the MICA-NKG2D interactionand prevent soluble MICA (sMICA)-induced downmodulation of NKG2D surfaceexpression; there are thereby useful to restore NKG2D expression in apatient. Such antibodies can also be used for their ability to block theNKG2D activation caused by the binding of MICA by NKG2D on a lymphocyteand will be particularly advantageous in the treatment of inflammationand autoimmune disorders. Other antibodies do not compete with NKG2D forbinding to MICA and will be advantageous in the treatment of cancer incases where NKG2D function on effector cells is desired to bemaintained. Optionally, the antibodies will not block oshedding of MICAfrom MICA-expressing tumor cell. Optionally, the antibodies bind theα1α2 domain of MICA (the portion of the MICA protein formed from the α1domain and α2 domain). Further provided are eptiopes on the α1α2 domainand α3 domain that are present across MICA alleles and can be targetedby antibodies for high binding affinity.

MICA (PERB11.1) refer to MHC class I polypeptide-related sequence A(See, e.g., UniProtKB/Swiss-Prot 029983), its gene and cDNA and its geneproduct, or naturally occurring variants thereof. Nomenclature of MICAgenes and proteins, together with reference to accession number ofsequence for different alleles are described in Frigoul A. and Lefranc,M-P. Recent Res. Devel. Human Genet., 3(2005): 95-145 ISBN:81-7736-244-5, the disclosure of which is incorporated herein byreference. MICA genes and protein sequence, including polymorphisms atthe protein and DNA level, are also available fromhttp://mhc-x.u-strasbg.fr/human.htm maintained by the laboratory of Dr.Bahram.

The amino acid sequences of MICA were first described in Bahram et al(1994) Proc. Nat. Acad. Sci. 91: 6259-6263 and Bahram et al. (1996)Immunogenetics 44:80-81, the disclosures of which are incorporatedherein by reference. The MICA gene is polymorphic, displaying an unusualdistribution of a number of variant amino acids in their extracellularα1, α2, and α3 domains. To further define the polymorphism of MICA,Petersdorf et al. (1999) examined its alleles among 275 individuals withcommon and rare HLA genotypes. The amino acid sequence of theextracellular α1, α2, and α3 domains of human MICA are shown in SEQ IDNOS 1-5. The full MICA sequence further comprises a leader sequence of23 amino acids, as well as a transmembrane domain and a cytoplasmicdomain. The amino acid sequence of extracellular α1, α2, and α3 domainsof selected human MICA alleles are shown in SEQ ID NOS 1-5. The aminoacid sequence of MICA*001 is shown in SEQ ID NO 1, corresponding toGenbank accession no. AAB41060. The amino acid sequence of human MICAallele MICA*004 is shown in SEQ ID NO 2, corresponding to Genbankaccession no. AAB41063. The amino acid sequence of human MICA alleleMICA*007 is shown in SEQ ID NO 3, corresponding to Genbank accession no.AAB41066. The amino acid sequence of human MICA allele MICA*008 is shownin SEQ ID NO 4, corresponding to Genbank accession no. AAB41067. Theamino acid sequence of human MICA allele MICA*019 is shown in SEQ ID NO5, corresponding to Genbank accession no. AAD27008. The amino acidsequence of human MICB is shown in SEQ ID NO 6, corresponding to Genbankaccession no. CAI18747.

The MICA gene encodes a protein that belongs to the MhcSF and to theIgSF. This protein is a transmembrane MHC-I-alpha-like (I-alpha-like)chain, which comprises three extracellular domains, two distal G-likedomains, G-alpha1-like (also referred to as “D1” or “α1”) andG-alpha2-like (also referred to as “D2” or “α2”), and a C-like-domain(also referred to as “D3” or “α3”) proximal to the cell membrane, andthree regions, a connecting-region, a transmembrane-region and acytoplasmic-region (labels according to the IMGT Scientific Chart of theIMGT (international ImMunoGeneTics Information System®), http://imgt.organd LeFranc et al. In Silico Biology, 2005; 5:45-60). The MICA matureprotein including leader, ECD, TM and CY domains, is made up of 360 to366 amino acids, the difference arising from a microsatellitepolymorphism in the transmembrane region. The α1, α2 and α3 can bedefined according to any suitable numbering system (e.g., the IMGTnumbering system). In one embodiment, the α1 domain comprises residuepositions 1 to 88 of the MICA polypeptide of SEQ ID NO 1; the α2 domaincomprises residue positions 89 to 181 of the MICA polypeptide of SEQ IDNO 1; and the α3 domain comprises residue positions 182 to 274 of theMICA polypeptide of SEQ ID NO 1. The α1 and α2 domains each comprise A,B, C and D strands, AB, BC and CD turns, and a helix. The α3 domaincomprises A, B, C, D, E, F and G strands, a BC loop, a CD strand, aDE-turn and an FG loop. The MICA protein is highly glycosylated witheight potential glycosylation sites, two in α1, one in α2 and five inthe α3 domain, including O-glycans (N-acetyllactosamine linked to serineor threonine) and/or N-glycans. While MICA is expressed constitutivelyin certain cells, low levels of MICA expression do not usually give riseto host immune cell attach. However, on MICA is upregulated on rapidlyproliferating cells such as tumor cells. MICA is the most highlyexpressed of all NKG2D ligands, and it has been found across a widerange of tumor types (e.g. carcinomas in general, bladder cancer,melanoma, lung cancer, hepatocellular cancer, glioblastoma, prostatecancer, hematological malignancies in general, acute myeloid leukemia,acute lymphatic leukemia, chronic myeloid leukemia and chronic lymphaticleukemia. Recently, Tsuboi et al. (2011) (EMBO 1-13) reported that theO-glycan branching enzyme, core2 β-1,6-N-acetylglucosaminyltransferase(C2GnT) is active in MICA-expressing tumor cells and that MICA fromtumor cells contains core2 O-glycan (an O-glycan comprising anN-acetylglucosamine branch connected to N-acetylgalactosamine).

Bauer et al Science 285: 727=729, 1999 provided a role for MICA as astress-inducible ligand for NKG2D. As used herein, “MICA” refers to anyMICA polypeptide, including any variant, derivative, or isoform of theMICA gene or encoded protein(s) to which they refer. The MICA gene ispolymorphic, displaying an unusual distribution of a number of variantamino acids in their extracellular alpha-1, alpha-2, and alpha-3domains. Various allelic variants have been reported for MICApolypeptides (e.g. MICA), each of these are encompassed by therespective terms, including, e.g., human MICA polypeptides MICA*001,MICA*002, MICA*004, MICA*005, MICA*006, MICA*007, MICA*008, MICA*009,MICA*010, MICA*011, MICA*012, MICA*013, MICA*014, MICA*015, MICA*016,MICA*017, MICA*018, MICA*019, MICA*020, MICA*022, MICA*023, MICA*024,MICA*025, MICA*026, MICA*027, MICA*028, MICA*029, MICA*030, MICA*031,MICA*032, MICA*033, MICA*034, MICA*035, MICA*036, MICA*037, MICA*038,MICA*039, MICA*040, MICA*041, MICA*042, MICA*043, MICA*044, MICA*045,MICA*046, MICA*047, MICA*048, MICA*049, MICA*050, MICA*051, MICA*052,MICA*053, MICA*054, MICA*055 and MICA*056. Also encompassed are anynucleic acid or protein sequences sharing one or more biologicalproperties or functions with wild type, full length MICA, respectively,and sharing at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or highernucleotide or amino acid identity.

As used herein, “hNKG2D” and, unless otherwise stated or contradicted bycontext, the terms “NKG2D,” “NKG2-D,” “CD314,” “D12S2489E,” “KLRK1,”“killer cell lectin-like receptor subfamily K, member 1,” or “KLRK1,”refer to a human killer cell activating receptor gene, its cDNA (e.g.,GenBank Accession No. NM_007360), and its gene product (GenBankAccession No. NP_031386), or naturally occurring variants thereof. In NKand T cells, hNKG2D can form heterodimers or higher order complexes withproteins such as DAP10 (GenBank Accession No. AAG29425, AAD50293). Anyactivity attributed herein to hNKG2D, e.g., cell activation, antibodyrecognition, etc., can also be attributed to hNKG2D in the form of aheterodimer such as hNKG2D-DAP10, or higher order complexes with thesetwo (and/or other) components.

The 3D structure of MICA in complex with NKG2D has been determined (see,e.g., Li et al., Nat. Immunol. 2001; 2:443-451; code 1hyr, and inIMGT/3Dstructure-DB (Kaas et al. Nucl. Acids Res. 2004; 32:D208-D210)).When MICA is in complex with a NKG2D homodimer, the residues 63 to 73(IGMT numbering) of MICA α2 are ordered, adding almost two turns ofhelix. The two monomers of NKG2D equally contribute to interactions withMICA, and seven positions in each NKG2D monomer interact with one of theMICA α1 or α2 helix domains.

The invention provides methods of using the antigen-binding compounds ofthe invention; for example, the invention provides a method forinhibiting cell proliferation or activity, for delivering a molecule toa cell (e.g. a toxic molecule, a detectable marker, etc.), fortargeting, identifying or purifying a cell, for depleting, killing oreliminating a cell, for reducing cell proliferation, the methodcomprising exposing a cell, such as a tumor cell which expresses a MICApolypeptide, to an antigen-binding compound of the invention that bindsa MICA polypeptide. It will be appreciated that for the purposes of thepresent invention, “cell proliferation” can refer to any aspect of thegrowth or proliferation of cells, e.g., cell growth, cell division, orany aspect of the cell cycle. The cell may be in cell culture (in vitro)or in a mammal (in vivo), e.g. a mammal suffering from a MICA-expressingpathology. The invention also provides a method for inducing the deathof a cell or inhibiting the proliferation or activity of a cell whichexpresses a MICA polypeptide, comprising exposing the cell to anantigen-binding compound that binds a MICA polypeptide linked to a toxicagent, in an amount effective to induce death and/or inhibit theproliferation of the cell. Thus, the invention provides a method fortreating a mammal suffering from a proliferative disease, and anycondition characterized by a pathogenic expansion of cells expressing ofa MICA polypeptide, the method comprising administering apharmaceutically effective amount of an antigen-binding compounddisclosed herein to the mammal, e.g. for the treatment of a cancer.

Definitions

As used in the specification, “a” or “an” may mean one or more. As usedin the claim(s), when used in conjunction with the word “comprising”,the words “a” or “an” may mean one or more than one. As used herein“another” may mean at least a second or more.

Where “comprising” is used, this can preferably be replaced by“consisting essentially of”, more preferably by “consisting of”.

Whenever within this whole specification “treatment of a proliferativedisease” or “treatment of a tumor”, or “treatment of cancer” or the likeis mentioned with reference to anti-MICA binding agent (e.g. antibody),there is meant: (a) method of treatment of a proliferative disease, saidmethod comprising the step of administering (for at least one treatment)an anti-MICA binding agent, (preferably in a pharmaceutically acceptablecarrier material) to a warm-blooded animal, especially a human, in needof such treatment, in a dose that allows for the treatment of saiddisease (a therapeutically effective amount), preferably in a dose(amount) as specified to be preferred hereinabove and herein below; (b)the use of an anti-MICA binding agent for the treatment of aproliferative disease, or an anti-MICA binding agent, for use in saidtreatment (especially in a human); (c) the use of an anti-MICA bindingagent, for the manufacture of a pharmaceutical preparation for thetreatment of a proliferative disease, a method of using an anti-MICAbinding agent for the manufacture of a pharmaceutical preparation forthe treatment of a proliferative disease, comprising admixing ananti-MICA binding agent with a pharmaceutically acceptable carrier, or apharmaceutical preparation comprising an effective dose of an anti-MICAbinding agent that is appropriate for the treatment of a proliferativedisease; or (d) any combination of a), b), and c), in accordance withthe subject matter allowable for patenting in a country where thisapplication is filed.

The terms “cancer” and “tumor” as used herein are defined as a newgrowth of cells or tissue comprising uncontrolled and progressivemultiplication. In a specific embodiment, upon a natural course thecancer is fatal. In specific embodiments, a cancer is invasive,metastatic, and/or anaplastic (loss of differentiation and oforientation to one another and to their axial framework).

The term “biopsy” as used herein is defined as removal of a tissue froman organ for the purpose of examination, such as to establish diagnosis.Examples of types of biopsies include by application of suction, such asthrough a needle attached to a syringe; by instrumental removal of afragment of tissue; by removal with appropriate instruments through anendoscope; by surgical excision, such as of the whole lesion; and thelike.

The term “antibody,” as used herein, refers to polyclonal and monoclonalantibodies. Depending on the type of constant domain in the heavychains, antibodies are assigned to one of five major classes: IgA, IgD,IgE, IgG, and IgM. Several of these are further divided into subclassesor isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. An exemplaryimmunoglobulin (antibody) structural unit comprises a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The N-terminus of each chain defines a variable region ofabout 100 to 110 or more amino acids that is primarily responsible forantigen recognition. The terms variable light chain (V_(L)) and variableheavy chain (V_(H)) refer to these light and heavy chains respectively.The heavy-chain constant domains that correspond to the differentclasses of immunoglobulins are termed “alpha,” “delta,” “epsilon,”“gamma” and “mu,” respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known. IgG and/or IgM are the preferred classes of antibodiesemployed in this invention, with IgG being particularly preferred,because they are the most common antibodies in the physiologicalsituation and because they are most easily made in a laboratory setting.Preferably the antibody of this invention is a monoclonal antibody.Particularly preferred are humanized, chimeric, human, orotherwise-human-suitable antibodies. “Antibodies” also includes anyfragment or derivative of any of the herein described antibodies.

The term “specifically binds to” means that an antibody can bindpreferably in a competitive binding assay to the binding partner, e.g.MICA, as assessed using either recombinant forms of the proteins,epitopes therein, or native proteins present on the surface of isolatedtarget cells. Competitive binding assays and other methods fordetermining specific binding are further described below and are wellknown in the art.

When an antibody is said to “compete with” a particular monoclonalantibody (e.g. 6E4, 20C6 or 16A8), it means that the antibody competeswith the monoclonal antibody in a binding assay using either recombinantMICA molecules or surface expressed MICA molecules. For example, if atest antibody reduces the binding of 6E4, 20C6 or 16A8 to a MICApolypeptide or MICA-expressing cell in a binding assay, the antibody issaid to “compete” respectively with 6E4, 20C6 or 16A8.

The term “affinity”, as used herein, means the strength of the bindingof an antibody to an epitope. The affinity of an antibody is given bythe dissociation constant K_(D), defined as [Ab]×[Ag]/[Ab-Ag], where[Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab]is the molar concentration of the unbound antibody and [Ag] is the molarconcentration of the unbound antigen. The affinity constant K_(a) isdefined by 1/Kd. Preferred methods for determining the affinity of mAbscan be found in Harlow, et al., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coliganet al., eds., Current Protocols in Immunology, Greene Publishing Assoc.and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol.92:589-601 (1983), which references are entirely incorporated herein byreference. One preferred and standard method well known in the art fordetermining the affinity of mAbs is the use of surface plasmon resonance(SPR) screening (such as by analysis with a BIAcore™ SPR analyticaldevice).

Within the context of this invention a “determinant” designates a siteof interaction or binding on a polypeptide.

The term “epitope” refers to an antigenic determinant, and is the areaor region on an antigen to which an antibody binds. A protein epitopemay comprise amino acid residues directly involved in the binding aswell as amino acid residues which are effectively blocked by thespecific antigen binding antibody or peptide, i.e., amino acid residueswithin the “footprint” of the antibody. It is the simplest form orsmallest structural area on a complex antigen molecule that can combinewith e.g., an antibody or a receptor. Epitopes can be linear orconformational/structural. The term “linear epitope” is defined as anepitope composed of amino acid residues that are contiguous on thelinear sequence of amino acids (primary structure). The term“conformational or structural epitope” is defined as an epitope composedof amino acid residues that are not all contiguous and thus representseparated parts of the linear sequence of amino acids that are broughtinto proximity to one another by folding of the molecule (secondary,tertiary and/or quaternary structures). A conformational epitope isdependent on the 3-dimensional structure. The term ‘conformational’ istherefore often used interchangeably with ‘structural’.

The term “depleting”, with respect to MICA-expressing cells means aprocess, method, or compound that can kill, eliminate, lyse or inducesuch killing, elimination or lysis, so as to negatively affect thenumber of MICA-expressing cells present in a sample or in a subject.

An “agent” or “compound” according to the present invention comprisessmall molecules, polypeptides, proteins, antibodies or antibodyfragments. Small molecules, in the context of the present invention,mean in one embodiment chemicals with molecular weight smaller than 1000Daltons, particularly smaller than 800 Daltons, more particularlysmaller than 500 Daltons. The term “therapeutic agent” refers to anagent that has biological activity. The term “anti-cancer agent” refersto an agent that has biological activity against cancer cells.

The term “human-suitable”, with respect to an antibody, refers to anyantibody, derivatized antibody, or antibody fragment that can be safelyused in humans for, e.g. the therapeutic methods described herein.Human-suitable antibodies include all types of humanized, chimeric, orfully human antibodies, or any antibodies in which at least a portion ofthe antibodies is derived from humans or otherwise modified so as toavoid the immune response that is generally provoked when nativenon-human antibodies are used.

For the purposes of the present invention, a “humanized” or “human”antibody refers to an antibody in which the constant and variableframework region of one or more human immunoglobulins is fused with thebinding region, e.g. the CDR, of an animal immunoglobulin. Suchantibodies are designed to maintain the binding specificity of thenon-human antibody from which the binding regions are derived, but toavoid an immune reaction against the non-human antibody. Such antibodiescan be obtained from transgenic mice or other animals that have been“engineered” to produce specific human antibodies in response toantigenic challenge (see, e.g., Green et al. (1994) Nature Genet 7:13;Lonberg et al. (1994) Nature 368:856; Taylor et al. (1994) Int Immun6:579, the entire teachings of which are herein incorporated byreference). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art (see, e.g., McCafferty et al. (1990)Nature 348:552-553). Human antibodies may also be generated by in vitroactivated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275,which are incorporated in their entirety by reference).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The terms “Fc domain,” “Fc portion,” and “Fc region” refer to aC-terminal fragment of an antibody heavy chain, e.g., from about aminoacid (aa) 230 to about aa 450 of human γ (gamma) heavy chain or itscounterpart sequence in other types of antibody heavy chains (e.g., α,δ, ε and μ for human antibodies), or a naturally occurring allotypethereof. Unless otherwise specified, the commonly accepted Kabat aminoacid numbering for immunoglobulins is used throughout this disclosure(see Kabat et al. (1991) Sequences of Protein of Immunological Interest,5th ed., United States Public Health Service, National Institute ofHealth, Bethesda, Md., also referred to as “Kabat EU”).

The term “antibody-dependent cell-mediated cytotoxicity” or “ADCC” is aterm well understood in the art, and refers to a cell-mediated reactionin which non-specific cytotoxic cells that express Fc receptors (FcRs)recognize bound antibody on a target cell and subsequently cause lysisof the target cell. Non-specific cytotoxic cells that mediate ADCCinclude natural killer (NK) cells, macrophages, monocytes, neutrophils,and eosinophils.

The term “complement-dependent cytotoxicity” or “CDC” is a term wellunderstood in the art, and refers to the ability of a molecule to lyse atarget in the presence of complement. The complement activation pathwayis initiated by the binding of the first component of the complementsystem (C1q) to a molecule (e.g. an antibody) complexed with a cognateantigen.

The term “shedding”, when referring to MICA, refers to release of asoluble extracellular domain (ECD) fragment of MICA from the cellsurface of a cell which expresses MICA. Such shedding may be caused byproteolytic cleavage (e.g. through the action of matrixmetalloproteinases (MMPs), e.g. ADAM10 and/or ADAM17) of cell surfaceMICA resulting in release of an ECD fragment from the cell surface, orthe soluble ECD or fragment thereof may be encoded by an alternatetranscript.

The terms “isolated”, “purified” or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (nonrecombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed ornot expressed at all.

As used herein, “NK cells” refers to a sub-population of lymphocytesthat is involved in non-conventional immunity. NK cells can beidentified by virtue of certain characteristics and biologicalproperties, such as the expression of specific surface antigensincluding CD56 and/or CD16 for human NK cells, the absence of thealpha/beta or gamma/delta TCR complex on the cell surface, the abilityto bind to and kill cells that fail to express “self” MHC/HLA antigensby the activation of specific cytolytic machinery, the ability to killtumor cells or other diseased cells that express a ligand for NKactivating receptors, and the ability to release protein moleculescalled cytokines that stimulate or inhibit the immune response. Any ofthese characteristics and activities can be used to identify NK cells,using methods well known in the art. Any subpopulation of NK cells willalso be encompassed by the term NK cells. Within the context of thisinvention “active” NK cells designate biologically active NK cells,including NK cells having the capacity of lysing target cells orenhancing the immune function of other cells. For instance, an “active”NK cell can be able to kill cells that express a ligand for anactivating NK receptor and/or fail to express MHC/HLA antigensrecognized by a KIR on the NK cell. NK cells can be obtained by varioustechniques known in the art, such as isolation from blood samples,cytapheresis, tissue or cell collections, etc. Useful protocols forassays involving NK cells can be found in Natural Killer Cells Protocols(edited by Campbell K S and Colonna M). Human Press. pp. 219-238 (2000).

As used herein, “T cells” refers to a sub-population of lymphocytes thatmature in the thymus, and which display, among other molecules T cellreceptors on their surface. T cells can be identified by virtue ofcertain characteristics and biological properties, such as theexpression of specific surface antigens including the TCR, CD4 or CD8,the ability of certain T cells to kill tumor or infected cells, theability of certain T cells to activate other cells of the immune system,and the ability to release protein molecules called cytokines thatstimulate or inhibit the immune response. Any of these characteristicsand activities can be used to identify T cells, using methods well knownin the art. Within the context of this invention, “active” or“activated” T or NK cells designate biologically active T or NK cells,more particularly T or NK cells having the capacity of cytolysis or ofstimulating an immune response by, e.g., secreting cytokines. Activecells can be detected in any of a number of well known methods,including functional assays and expression-based assays such as theexpression of cytokines.

Within the context of this invention, the term antibody that “binds” apolypeptide or epitope designates an antibody that binds saiddeterminant with specificity and/or affinity.

Antibodies

The antibodies of the present invention are antibodies that bind humanMICA across different alleles. In one embodiment, the antibodies bind toa MICA polypeptide (an anti-MICA antibody) without substantiallyblocking the interaction of MICA with NKG2D (e.g., the interaction ofsurface MICA on tumor cells with surface NKG2D on effector cells). Inanother embodiment, the antibodies bind to a MICA polypeptide (ananti-MICA antibody) and inhibit the interaction of MICA with NKG2D;preferably the antibodies inhibit downmodulation of NKG2D on the surfaceof immune cells caused by sMICA. In one embodiment, the antibodies bindto a MICA polypeptide on the surface of a cell without substantiallyblocking shedding of MICA from the cell surface (e.g. of tumor cells).In one embodiment, the antibodies bind a 1 and/or α2 domains of MICA. Inone embodiment, the antibodies bind the α3 domain of MICA. In oneembodiment, the antibodies have an affinity for human MICA alleles *001;*004 and *008, optionally further *007 and/or *019, optionallycharacterized by a Kd of less than 10⁻⁹ M, preferably less than 10⁻¹⁰M.

In one embodiment, the antibody competes for binding to the MICApolypeptide with any one or more of antibodies 6E4, 20C6, 16A8, 9C10,19E9, 12A10, 10A7, 18E8, 10F3, 15F9 and 14B4. Preferably the antibodyrecognizes, binds to, or has immunospecificity for substantially oressentially the same, or the same, epitope or “epitopic site” on a MICApolypeptide.

Antibody Epitopes

In another embodiment, the antibodies bind substantially the sameepitope as antibody 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8,10F3, 15F9 and 14B4. In another embodiment, the antibodies at leastpartially overlaps, or includes at least one residue in the segmentcorresponding to residues 1-88, residues 89-181, or residues 182-274 ofa MICA polypeptide comprising an amino acid sequence of SEQ ID NOS: 1 to5. In one embodiment, all key residues of the epitope is in a segmentcorresponding to residues 1-88, residues 89-181, or residues 182-274 ofa MICA polypeptide comprising an amino acid sequence of SEQ ID NOS: 1 to5. In one embodiment, an antibody binds an epitope spanning the junctionof (a) the α1 and/or α2 domain and (b) the α3 domain, wherein all keyresidues of the epitope is in a segment corresponding to residues 1-181(e.g., residues 1-88 (optionally 1-85) or 89-181 (optionally 86-181)) ofa MICA polypeptide comprising an amino acid sequence of SEQ ID NOS: 1 to5. In one embodiment, the antibodies bind an epitope comprising 1, 2, 3,4, 5, 6, 7 or more residues in the segment corresponding to residues1-88 (optionally 1-85) or residues 89-181 (optionally 86-181) of a MICApolypeptide comprising an amino acid sequence of SEQ ID NOS: 1 to 5.Preferably the residues bound by the antibody are present on the surfaceof the of the MICA polypeptide, e.g. in a MICA polypeptide expressed onthe surface of a cell.

In one embodiment, an antibody binds an epitope comprising 1, 2, 3, 4,5, 6, 7 or more residues selected from the group consisting of R6, N8,Q48, W49, E51, D52, V53, L54, N56, K57, T58, R61, R64, K81, D82, Q83,K84, E97, H99, E100, D101, N102, S103, T104, R105, H109, Y111, D113,E115, L116, N121, E123, T124, E126, Q131, S132, S133, R134, Q136, T137,M140, N141, R143, N144, L178, R179, R180, S224, H225, D226, T227, Q228,Q229, W230 and D232 (with reference to a MICA of any of SEQ ID NOS 1-5).

In one embodiment, an antibody binds an epitope comprising:

-   -   (a) one or more residues selected from the group consisting of        R6 and N8;    -   (b) one or more residues selected from the group consisting of        N56, K57, T58;    -   (c) one or more residues selected from the group consisting of        R61 and R64;    -   (d) one or more residues selected from the group consisting of        K81, D82;    -   (e) one or more residues selected from the group consisting of        Q83, K84;    -   (f) one or more residues selected from the group consisting of        E97, H99;    -   (g) one or more residues selected from the group consisting of        E100, D101, N102;    -   (h) one or more residues selected from the group consisting of        S103, T104, R105;    -   (i) one or more residues selected from the group consisting of        D113, E115;    -   (j) one or more residues selected from the group consisting of        N121, E123;    -   (k) one or more residues selected from the group consisting of        T124 and E126;    -   (l) one or more residues selected from the group consisting of        H109, Y111, L116;    -   (m) one or more residues selected from the group consisting of        Q131, S132, Q136;    -   (n) one or more residues selected from the group consisting of        S133, R134, T137; or    -   (o) one or more residues selected from the group consisting of        M140, N141, R143 and N144;    -   (p) one or more residues selected from the group consisting of        S224, H225 and D226;    -   (q) one or more residues selected from the group consisting of        T227, Q228 and Q229; and/or    -   (r) one or more residues selected from the group consisting of        W230 and D232.        In one embodiment, an antibody binds an epitope comprising or        any combination of 2, 3 or 4 of (a) to (r).

In one embodiment, an antibody binds an epitope comprising 1, 2, 3, 4,5, or 6 or more residues selected from the group consisting of Q48, W49,E51, D52, V53 and L54.

In one embodiment, an antibody binds an epitope comprising 1, 2, 3, 4,5, or 6 or more residues selected from the group consisting of N56, K57,T58, R61 and R64.

In one embodiment, an antibody binds an epitope comprising 1, 2, 3, 4,5, or 6 or more residues selected from the group consisting of K81, D82,Q83, K84, H109, Y111, D113, L116, S133, R134, T137, M140, N141, R143 andN144.

In one embodiment, an antibody binds an epitope comprising 1, 2, 3, 4,5, or 6 or more residues selected from the group consisting of K81, D82,083, K84, H109, Y111, D113, L116, Q131, S132, Q136, M140, N141, R143 andN144.

In one embodiment, an antibody binds an epitope comprising 1, 2, 3, 4,5, or 6 or more residues selected from the group consisting of E100,D101, N102, S103, T104, R105, N121, E123, T124 and E126.

In one embodiment, the antibodies bind an epitope comprising 1, 2, 3, 4,5, or 6 or more residues selected from the group consisting of R6, N8,E97, H99, E100, D101, N102, S103, 1104, R105, E115, L178, R179 and R180.

In one embodiment, an antibody binds an epitope comprising 1, 2, 3, 4,5, or 6 or more residues selected from the group consisting of S224,H225, D226, T227, Q228, Q229, W230 and D232. In one embodiment, anantibody binds an epitope comprising 1, 2, 3, 4, 5, or 6 or moreresidues selected from the group consisting of T227, Q228 and Q229.

In one embodiment, an antibody binds an epitope comprising:

-   -   (a) 1 or more residues selected from the group consisting of        K81, D82, and 1 or more residues selected from the group        consisting of Q83, K84;    -   (b) 1, 2, 3, 4 or more residues selected from the group        consisting of K81, D82, Q83, K84, and 1, 2, or 3 residues        selected from the group consisting of H109, Y111, L116;    -   (c) residue D113, and 1, 2, 3 or 4 residues selected from the        group consisting of K81, D82, Q83, K84;    -   (d) 1, 2, 3, 4 or more residues selected from the group        consisting of K81, D82, Q83, K84, and 1, 2, or 3 residues        selected from the group consisting of Q131, S132, Q136;    -   (e) 1, 2, 3, 4 or more residues selected from the group        consisting of K81, D82, Q83, K84, and 1, 2, or 3 residues        selected from the group consisting of S133, R134, T137;    -   (f) 1, 2, 3, 4 or more residues selected from the group        consisting of K81, D82, Q83, K84, and 1, 2, or 3 residues        selected from the group consisting of M140, N141, R143 and N144;    -   (g) 1, 2, 3, 4 or more residues selected from the group        consisting of K81, D82, Q83, K84; 1, 2 or 3 residues selected        from the group consisting of H109, Y111, L116; optionally D113;        and 1, 2 or 3 residues selected from the group consisting of        Q131, S132, Q136;    -   (h) 1, 2, 3, 4 or more residues selected from the group        consisting of K81, D82, Q83, K84; 1, 2 or 3 residues selected        from the group consisting of H109, Y111, L116; optionally D113;        and 1, 2 or 3 residues selected from the group consisting of        S133, R134, T137;    -   (i) 1, 2, 3, 4 or more residues selected from the group        consisting of K81, D82, Q83, K84; 1, 2 or 3 residues selected        from the group consisting of H109, Y111, L116; optionally D113;        and −1, 2, 3, 4 or more residues selected from the group        consisting of M140, N141, R143 and N144;    -   (j) 1, 2, 3, 4 or more residues selected from the group        consisting of K81, D82, Q83, K84; 1, 2 or 3 residues selected        from the group consisting of H109, Y111, L116; optionally D113;        1, 2 or 3 residues selected from the group consisting of S133,        R134, T137; and 1, 2, 3 or 4 residues selected from the group        consisting of M140, N141, R143 and N144;    -   (k) 1 or more residues selected from the group consisting of R6,        N8, and 1, 2, 3, 4 or more residues selected from the group        consisting of E100, D101, N102, S103, T104, R105;    -   (l) 1, 2, 3 or 4 residues selected from the group consisting of        N121, E123, T124 and E126, and 1, 2, 3, 4 or more residues        selected from the group consisting of E100, D101, N102, S103,        1104, R105; or    -   (m) 1, 2, or 3 residues selected from the group consisting of        S224, H225 and D226, 1, 2, or 3 residues selected from the group        consisting of T227, Q228 and Q229, and one or two residues        selected from the group consisting of W230 and D232.

Optionally, the epitope of an antibody of the invention may be entirelywithin the α1 and/or α2 domains of MICA. Optionally, further, theantibodies can be characterized as not substantially binding to the α3domain region required for MICA shedding.

In one embodiment, the antibodies of the invention bind one or moreamino acids present on the surface of the MICA polypeptide alleles *001,*004 and *008 (and optionally further *007 and *019).

Binding of anti-MICA antibody to cells transfected with the MICA mutantscan be measured and compared to the ability of anti-MICA antibody tobind wild-type MICA polypeptide (e.g., any one or more of SEQ ID NOS: 1to 5). A reduction in binding between an anti-MICA antibody and a mutantMICA polypeptide means that there is a reduction in binding affinity(e.g., as measured by known methods such FACS testing of cellsexpressing a particular mutant, or by Biacore testing of binding tomutant polypeptides) and/or a reduction in the total binding capacity ofthe anti-MICA antibody (e.g., as evidenced by a decrease in Bmax in aplot of anti-MICA antibody concentration versus polypeptideconcentration). A significant reduction in binding indicates that themutated residue is directly involved in binding to the anti-MICAantibody or is in close proximity to the binding protein when theanti-MICA antibody is bound to MICA.

In some embodiments, a significant reduction in binding means that thebinding affinity and/or capacity between an anti-MICA antibody and amutant MICA polypeptide is reduced by greater than 40%, greater than50%, greater than 55%, greater than 60%, greater than 65%, greater than70%, greater than 75%, greater than 80%, greater than 85%, greater than90% or greater than 95% relative to binding between the antibody and awild type MICA polypeptide. In certain embodiments, binding is reducedbelow detectable limits. In some embodiments, a significant reduction inbinding is evidenced when binding of an anti-MICA antibody to a mutantMICA polypeptide is less than 50% (e.g., less than 45%, 40%, 35%, 30%,25%, 20%, 15% or 10%) of the binding observed between the anti-MICAantibody and a wild-type MICA polypeptide.

In some embodiments, anti-MICA antibodies are provided that exhibitsignificantly lower binding for a mutant MICA polypeptide in which aresidue in a segment corresponding to residues 1-88 (optionally 1-85),residues 89-181 (optionally 86-181), or residues 182-274 (or asubsequence thereof) in a wild-type MICA polypeptide (e.g., comprising asequence of SEQ ID NOS: 1 to 5) is substituted with a different aminoacid.

In some embodiments, anti-MICA antibodies are provided that exhibitsignificantly lower binding for a mutant MICA polypeptide in which aresidue in a segment corresponding to residues 1-88 (optionally 1-85),residues 89-181 (optionally 86-181), or residues 182-274 (or asubsequence thereof) in a wild-type MICA polypeptide (e.g., comprising asequence of SEQ ID NOS: 1 to 5) is substituted with a different aminoacid.

In some embodiments, anti-MICA antibodies are provided that exhibitsignificantly lower binding for a mutant MICA polypeptide in which aresidue selected from the group consisting of R6, N8, Q48, W49, E51,D52, V53, L54, N56, K57, T58, R61, R64, K81, D82, Q83, K84, E97, H99,E100, D101, N102, S103, T104, R105, H109, Y111, D113, E115, L116, N121,E123, T124, E126, Q131, S132, S133, R134, Q136, T137, M140, N141, R143,N144, L178, R179, R180, S224, H225, D226, T227, Q228, Q229, W230 andD232 is substituted with a different amino acid, compared to a wild-typeMICA polypeptide.

In some embodiments, anti-MICA antibodies are provided that exhibitsignificantly lower binding for a mutant MICA polypeptide in which:

-   -   (a) 1, 2, 3, 4 or more residues selected from the group        consisting of Q48, W49, E51, D52, V53 and L54;    -   (b) 1, 2, 3, 4 or more residues selected from the group        consisting of N56, K57, T58, R61 and R64;    -   (c) 1, 2, 3, 4 or more residues selected from the group        consisting of K81, D82, Q83, K84, H109, Y111, D113, L116, S133,        R134, T137, M140, N141, R143 and N144;    -   (d) 1, 2, 3, 4 or more residues selected from the group        consisting of K81, D82, Q83, K84, H109, Y111, D113, L116, 0131,        S132, Q136, M140, N141, R143 and N144;    -   (e) 1, 2, 3, 4 or more residues selected from the group        consisting of E100, D101, N102, S103, T104, R105, N121, E123,        T124 and E126;    -   (f) 1, 2, 3, 4 or more residues selected from the group        consisting of R6, N8, E97, H99, E100, D101, N102, S103, T104,        R105, E115, L178, R179 and R180; or    -   (g) 1, 2, 3, 4 or more residues selected from the group        consisting of S224, H225, D226, T227, 0228, Q229, W230 and D232,    -   is substituted with a different amino acid, compared to a        wild-type MICA polypeptide.

In some embodiments, anti-MICA antibodies are provided that exhibitsignificantly lower binding for a mutant MICA polypeptide in which:

-   -   (a) a residue selected from the group consisting of R6 and N8;    -   (b) a residue selected from the group consisting of N56, K57,        T58;    -   (c) a residue selected from the group consisting of R61 and R64;    -   (d) a residue selected from the group consisting of K81, D82;    -   (e) a residue selected from the group consisting of Q83, K84;    -   (f) a residue selected from the group consisting of E97, H99;    -   (g) a residue selected from the group consisting of E100, D101,        N102;    -   (h) a residue selected from the group consisting of S103, T104,        R105;    -   (i) a residue selected from the group consisting of D113, E115;    -   (j) a residue selected from the group consisting of N121, E123;    -   (k) a residue selected from the group consisting of T124 and        E126;    -   (l) a residue selected from the group consisting of H109, Y111,        L116;    -   (m) a residue selected from the group consisting of Q131, S132,        Q136;    -   (n) a residue selected from the group consisting of S133, R134,        I137;    -   (o) a residue selected from the group consisting of M140, N141,        R143 and N144;    -   (p) a residue selected from the group consisting of S224, H225        and D226;    -   (q) a residue selected from the group consisting of T227, Q228        and Q229; and/or    -   (r) a residue selected from the group consisting of W230 and        D232, is substituted with a different amino acid, compared to a        wild-type MICA polypeptide.

In any embodiments a R6, N8, Q48, W49, E51, D52, V53, L54, N56, K57,T58, R61, R64, K81, D82, Q83, K84, E97, H99, E100, D101, N102, S103,T104, R105, H109, Y111, D113, E115, L116, N121, E123, T124, E126, Q131,S132, S133, R134, Q136, T137, M140, N141, R143, N144, L178, R179, R180,S194, E195, N197, S224, H225, D226, T227, Q228, Q229, W230 or D232substitution may be specified as being a R6A, N8A, W14A, Q48A, W49S,E51S, D52A, V53S, L54A, N56A, K57S, T58A, R61A, R64A, K81A, D82A, Q83A,K84A, E85A, E97A, H99A, E100A, D101S, N102A, S103A, T104S, R105A, H109A,Y111A, D113A, E115A, L116A, N121A, E123S, T124A, E126A, Q131A, S132A,S133A, R134S, Q136S, T137A, M140S, N141A, R143S, N144A, L178A, R179S,R180A, S224A, H225S, D226A, T227A, Q228S, Q229A, W230A or D232Asubstitution, respectively.

In some embodiments, anti-MICA antibodies are characterized by notexhibiting significantly lower binding for a mutant MICA polypeptide(e.g. a mutant of any one of mutants 1-61 of Table D) or to apolypeptide having a mutated residue of any one of mutants 1-61 of TableD, compared to a wild-type MICA polypeptide (other than the mutant(s) orresidue(s) to which a particular anti-MICA antibody has significantlylower binding as shown in Example 4).

Producing Anti-MICA Antibodies

The antibodies of this invention may be produced by a variety oftechniques known in the art. Typically, they are produced byimmunization of a non-human animal, preferably a mouse, with animmunogen comprising a MICA polypeptide, preferably a human MICApolypeptide. The MICA polypeptide may comprise the full length sequenceof a human MICA polypeptide, or a fragment or derivative thereof,typically an immunogenic fragment, i.e., a portion of the polypeptidecomprising an epitope exposed on the surface of cells expressing a MICApolypeptide, preferably the epitope recognized by the 6E4, 20C6, 16A8,9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9 or 14B4 antibody. Suchfragments typically contain at least about 7 consecutive amino acids ofthe mature polypeptide sequence, even more preferably at least about 10consecutive amino acids thereof. Fragments typically are essentiallyderived from the extra-cellular domain of the receptor.

In a preferred embodiment, the immunogen comprises a wild-type humanMICA polypeptide in a lipid membrane, typically at the surface of acell. In a specific embodiment, the immunogen comprises intact cells,particularly intact human cells, optionally treated or lysed. In anotherpreferred embodiment, the polypeptide is a recombinant MICA polypeptide.In a specific embodiment, the immunogen comprises intact tumor cells.

The step of immunizing a non-human mammal with an antigen may be carriedout in any manner well known in the art for stimulating the productionof antibodies in a mouse (see, for example, E. Harlow and D. Lane,Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1988), the entire disclosure of which isherein incorporated by reference). The immunogen is suspended ordissolved in a buffer, optionally with an adjuvant, such as complete orincomplete Freund's adjuvant. Methods for determining the amount ofimmunogen, types of buffers and amounts of adjuvant are well known tothose of skill in the art and are not limiting in any way on the presentinvention. These parameters may be different for different immunogens,but are easily elucidated.

Similarly, the location and frequency of immunization sufficient tostimulate the production of antibodies is also well known in the art. Ina typical immunization protocol, the non-human animals are injectedintraperitoneally with antigen on day 1 and again about a week later.This is followed by recall injections of the antigen around day 20,optionally with an adjuvant such as incomplete Freund's adjuvant. Therecall injections are performed intravenously and may be repeated forseveral consecutive days. This is followed by a booster injection at day40, either intravenously or intraperitoneally, typically withoutadjuvant. This protocol results in the production of antigen-specificantibody-producing B cells after about 40 days. Other protocols may alsobe used as long as they result in the production of B cells expressingan antibody directed to the antigen used in immunization.

For polyclonal antibody preparation, serum is obtained from an immunizednon-human animal and the antibodies present therein isolated bywell-known techniques. The serum may be affinity purified using any ofthe immunogens set forth above linked to a solid support so as to obtainantibodies that react with MICA polypeptides.

In an alternate embodiment, lymphocytes from a non-immunized non-humanmammal are isolated, grown in vitro, and then exposed to the immunogenin cell culture. The lymphocytes are then harvested and the fusion stepdescribed below is carried out.

For preferred monoclonal antibodies, the next step is the isolation ofsplenocytes from the immunized non-human mammal and the subsequentfusion of those splenocytes with an immortalized cell in order to forman antibody-producing hybridoma. The isolation of splenocytes from anon-human mammal is well-known in the art and typically involvesremoving the spleen from an anesthetized non-human mammal, cutting itinto small pieces and squeezing the splenocytes from the splenic capsulethrough a nylon mesh of a cell strainer into an appropriate buffer so asto produce a single cell suspension. The cells are washed, centrifugedand resuspended in a buffer that lyses any red blood cells. The solutionis again centrifuged and remaining lymphocytes in the pellet are finallyresuspended in fresh buffer.

Once isolated and present in single cell suspension, the lymphocytes canbe fused to an immortal cell line. This is typically a mouse myelomacell line, although many other immortal cell lines useful for creatinghybridomas are known in the art. Preferred murine myeloma lines include,but are not limited to, those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, U.S.A., X63 Ag8653 and SP-2 cells available from the AmericanType Culture Collection, Rockville, Md. U.S.A. The fusion is effectedusing polyethylene glycol or the like. The resulting hybridomas are thengrown in selective media that contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Hybridomas are typically grown on a feeder layer of macrophages. Themacrophages are preferably from littermates of the non-human mammal usedto isolate splenocytes and are typically primed with incomplete Freund'sadjuvant or the like several days before plating the hybridomas. Fusionmethods are described in Goding, “Monoclonal Antibodies: Principles andPractice,” pp. 59-103 (Academic Press, 1986), the disclosure of which isherein incorporated by reference.

The cells are allowed to grow in the selection media for sufficient timefor colony formation and antibody production. This is usually betweenabout 7 and about 14 days.

The hybridoma colonies are then assayed for the production of antibodiesthat specifically bind to MICA polypeptide gene products, optionally theepitope specifically recognized by antibody 6E4, 20C6, 16A8, 9C10, 19E9,12A10, 10A7, 18E8, 10F3, 15F9 or 1484. The assay is typically acolorimetric ELISA-type assay, although any assay may be employed thatcan be adapted to the wells that the hybridomas are grown in. Otherassays include radioimmunoassays or fluorescence activated cell sorting.The wells positive for the desired antibody production are examined todetermine if one or more distinct colonies are present. If more than onecolony is present, the cells may be re-cloned and grown to ensure thatonly a single cell has given rise to the colony producing the desiredantibody.

Hybridomas that are confirmed to produce a monoclonal antibody of thisinvention can be grown up in larger amounts in an appropriate medium,such as DMEM or RPMI-1640. Alternatively, the hybridoma cells can begrown in vivo as ascites tumors in an animal.

After sufficient growth to produce the desired monoclonal antibody, thegrowth media containing monoclonal antibody (or the ascites fluid) isseparated away from the cells and the monoclonal antibody presenttherein is purified. Purification is typically achieved by gelelectrophoresis, dialysis, chromatography using protein A or proteinG-Sepharose, or an anti-mouse Ig linked to a solid support such asagarose or Sepharose beads (all described, for example, in the AntibodyPurification Handbook, Biosciences, publication No. 18-1037-46, EditionAC, the disclosure of which is hereby incorporated by reference). Thebound antibody is typically eluted from protein A/protein G columns byusing low pH buffers (glycine or acetate buffers of pH 3.0 or less) withimmediate neutralization of antibody-containing fractions. Thesefractions are pooled, dialyzed, and concentrated as needed.

Positive wells with a single apparent colony are typically re-cloned andre-assayed to insure only one monoclonal antibody is being detected andproduced.

Antibodies may also be produced by selection of combinatorial librariesof immunoglobulins, as disclosed for instance in (Ward et al. Nature,341 (1989) p. 544, the entire disclosure of which is herein incorporatedby reference).

The identification of one or more antibodies that bind(s) to MICA,particularly substantially or essentially the same epitope as monoclonalantibody 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9 or14B4, can be readily determined using any one of a variety ofimmunological screening assays in which antibody competition can beassessed. Many such assays are routinely practiced and are well known inthe art (see, e. g., U.S. Pat. No. 5,660,827, issued Aug. 26, 1997,which is specifically incorporated herein by reference). It will beunderstood that actually determining the epitope to which an antibodydescribed herein binds is not in any way required to identify anantibody that binds to the same or substantially the same epitope as themonoclonal antibody described herein.

For example, where the test antibodies to be examined are obtained fromdifferent source animals, or are even of a different Ig isotype, asimple competition assay may be employed in which the control (6E4,20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9 or 14B4, forexample) and test antibodies are admixed (or pre-adsorbed) and appliedto a sample containing MICA polypeptides. Protocols based upon westernblotting and the use of BIACORE analysis are suitable for use in suchcompetition studies.

In certain embodiments, one pre-mixes the control antibodies (6E4, 20C6or 16A8, for example) with varying amounts of the test antibodies (e.g.,about 1:10 or about 1:100) for a period of time prior to applying to theMICA antigen sample. In other embodiments, the control and varyingamounts of test antibodies can simply be admixed during exposure to theMICA antigen sample. As long as one can distinguish bound from freeantibodies (e. g., by using separation or washing techniques toeliminate unbound antibodies) and 6E4, 20C6 or 16A8 from the testantibodies (e. g., by using species-specific or isotype-specificsecondary antibodies or by specifically labeling 6E4, 20C6 or 16A8 witha detectable label) one can determine if the test antibodies reduce thebinding of 6E4, 20C6 or 16A8 to the antigens, indicating that the testantibody recognizes substantially the same epitope as 6E4, 20C6 or 16A8.The binding of the (labeled) control antibodies in the absence of acompletely irrelevant antibody can serve as the control high value. Thecontrol low value can be obtained by incubating the labeled (6E4, 20C6or 16A8) antibodies with unlabelled antibodies of exactly the same type(6E4, 20C6 or 16A8), where competition would occur and reduce binding ofthe labeled antibodies. In a test assay, a significant reduction inlabeled antibody reactivity in the presence of a test antibody isindicative of a test antibody that recognizes substantially the sameepitope, i.e., one that “cross-reacts” or competes with the labeled(6E4, 20C6 or 16A8) antibody. Any test antibody that reduces the bindingof 6E4, 20C6 or 16A8 to MICA antigens by at least about 50%, such as atleast about 60%, or more preferably at least about 80% or 90% (e. g.,about 65-100%), at any ratio of 6E4, 20C6 or 16A8:test antibody betweenabout 1:10 and about 1:100 is considered to be an antibody that binds tosubstantially the same epitope or determinant as 6E4, 20C6 or 16A8.Preferably, such test antibody will reduce the binding of 6E4, 20C6 or16A8 to the MICA antigen by at least about 90% (e.g., about 95%).

Competition can also be assessed by, for example, a flow cytometry test.In such a test, cells bearing a given MICA polypeptide can be incubatedfirst with 6E4, 20C6 or 16A8, for example, and then with the testantibody labeled with a fluorochrome or biotin. The antibody is said tocompete with 6E4, 20C6 or 16A8 if the binding obtained uponpreincubation with a saturating amount of 6E4, 20C6 or 16A8 is about80%, preferably about 50%, about 40% or less (e.g., about 30%, 20% or10%) of the binding (as measured by mean of fluorescence) obtained bythe antibody without preincubation with 6E4, 20C6 or 16A8.Alternatively, an antibody is said to compete with 6E4, 20C6 or 16A8 ifthe binding obtained with a labeled 6E4, 20C6 or 16A8 antibody (by afluorochrome or biotin) on cells preincubated with a saturating amountof test antibody is about 80%, preferably about 50%, about 40%, or less(e. g., about 30%, 20% or 10%) of the binding obtained withoutpreincubation with the test antibody.

A simple competition assay in which a test antibody is pre-adsorbed andapplied at saturating concentration to a surface onto which a MICAantigen is immobilized may also be employed. The surface in the simplecompetition assay is preferably a BIACORE chip (or other media suitablefor surface plasmon resonance analysis). The control antibody (e.g.,6E4, 20C6 or 16A8) is then brought into contact with the surface at aMICA-saturating concentration and the MICA and surface binding of thecontrol antibody is measured. This binding of the control antibody iscompared with the binding of the control antibody to the MICA-containingsurface in the absence of test antibody. In a test assay, a significantreduction in binding of the MICA-containing surface by the controlantibody in the presence of a test antibody indicates that the testantibody recognizes substantially the same epitope as the controlantibody such that the test antibody “cross-reacts” with the controlantibody. Any test antibody that reduces the binding of control (such as6E4, 20C6 or 16A8) antibody to a MICA antigen by at least about 30% ormore, preferably about 40%, can be considered to be an antibody thatbinds to substantially the same epitope or determinant as a control(e.g., 6E4, 20C6 or 16A8). Preferably, such a test antibody will reducethe binding of the control antibody (e.g., 6E4, 20C6 or 16A8) to theMICA antigen by at least about 50% (e. g., at least about 60%, at leastabout 70%, or more). It will be appreciated that the order of controland test antibodies can be reversed: that is, the control antibody canbe first bound to the surface and the test antibody is brought intocontact with the surface thereafter in a competition assay. Preferably,the antibody having higher affinity for the MICA antigen is bound to thesurface first, as it will be expected that the decrease in binding seenfor the second antibody (assuming the antibodies are cross-reacting)will be of greater magnitude. Further examples of such assays areprovided in, e.g., Saunal (1995) J. Immunol. Methods 183: 33-41, thedisclosure of which is incorporated herein by reference.

Preferably, monoclonal antibodies that recognize a MICA epitope willreact with an epitope that is present on a substantial percentage of oreven all relevant MICA alleles. In one aspect, the anti-MICA antibodiesof the invention bind MICA*004 and *008, optionally further MICA *001,*007 and/or *0019.

In preferred embodiments, the antibodies will bind to MICA-expressingcells from an individual or individuals with a disease characterized byexpression of MICA-positive cells, i.e. an individual that is acandidate for treatment with one of the herein-described methods usingan anti-MICA antibody of the invention. Accordingly, once an antibodythat specifically recognizes MICA on cells is obtained, it can be testedfor its ability to bind to MICA-positive cells (e.g. cancer cells). Inparticular, prior to treating a patient with one of the presentantibodies, it will be beneficial to test the ability of the antibody tobind malignant cells taken from the patient, e.g. in a blood sample ortumor biopsy, to maximize the likelihood that the therapy will bebeneficial in the patient.

In one embodiment, the antibodies of the invention are validated in animmunoassay to test their ability to bind to MICA-expressing cells, e.g.malignant cells. For example, a tumor biopsy is performed and tumorcells are collected. The ability of a given antibody to bind to thecells is then assessed using standard methods well known to those in theart. Antibodies that are found to bind to a substantial proportion(e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80% or more) of cells known toexpress MICA, e.g. tumor cells, from a significant percentage ofindividuals or patients (e.g., 5%, 10%, 20%, 30%, 40%, 50% or more) aresuitable for use in the present invention, both for diagnostic purposesto determine the presence or level of malignant cells in a patient orfor use in the herein-described therapeutic methods, e.g., for use toincrease or decrease malignant cell number or activity. To assess thebinding of the antibodies to the cells, the antibodies can either bedirectly or indirectly labeled. When indirectly labeled, a secondary,labeled antibody is typically added.

Determination of whether an antibody binds within an epitope region canbe carried out in ways known to the person skilled in the art. As oneexample of such mapping/characterization methods, an epitope region foran anti-MICA antibody may be determined by epitope “foot-printing” usingchemical modification of the exposed amines/carboxyls in the MICAprotein. One specific example of such a foot-printing technique is theuse of HXMS (hydrogen-deuterium exchange detected by mass spectrometry)wherein a hydrogen/deuterium exchange of receptor and ligand proteinamide protons, binding, and back exchange occurs, wherein the backboneamide groups participating in protein binding are protected from backexchange and therefore will remain deuterated. Relevant regions can beidentified at this point by peptic proteolysis, fast microborehigh-performance liquid chromatography separation, and/or electrosprayionization mass spectrometry. See, e. g., Ehring H, AnalyticalBiochemistry, Vol. 267 (2) pp. 252-259 (1999) Engen, J. R. and Smith, D.L. (2001) Anal. Chem. 73, 256A-265A. Another example of a suitableepitope identification technique is nuclear magnetic resonance epitopemapping (NMR), where typically the position of the signals intwo-dimensional NMR spectra of the free antigen and the antigencomplexed with the antigen binding peptide, such as an antibody, arecompared. The antigen typically is selectively isotopically labeled with15N so that only signals corresponding to the antigen and no signalsfrom the antigen binding peptide are seen in the NMR-spectrum. Antigensignals originating from amino acids involved in the interaction withthe antigen binding peptide typically will shift position in thespectrum of the complex compared to the spectrum of the free antigen,and the amino acids involved in the binding can be identified that way.See, e. g., Ernst Schering Res Found Workshop. 2004; (44): 149-67; Huanget al., Journal of Molecular Biology, Vol. 281 (1) pp. 61-67 (1998); andSaito and Patterson, Methods. 1996 June; 9 (3): 516-24.

Epitope mapping/characterization also can be performed using massspectrometry methods. See, e.g., Downard, J Mass Spectrom. 2000 Apr.; 35(4): 493-503 and Kiselar and Downard, Anal Chem. 1999 May 1; 71 (9):1792-1801. Protease digestion techniques also can be useful in thecontext of epitope mapping and identification. Antigenicdeterminant-relevant regions/sequences can be determined by proteasedigestion, e.g. by using trypsin in a ratio of about 1:50 to MICA or o/ndigestion at and pH 7-8, followed by mass spectrometry (MS) analysis forpeptide identification. The peptides protected from trypsin cleavage bythe anti-MICA binder can subsequently be identified by comparison ofsamples subjected to trypsin digestion and samples incubated withantibody and then subjected to digestion by e.g. trypsin (therebyrevealing a footprint for the binder). Other enzymes like chymotrypsin,pepsin, etc., also or alternatively can be used in similar epitopecharacterization methods. Moreover, enzymatic digestion can provide aquick method for analyzing whether a potential antigenic determinantsequence is within a region of the MICA polypeptide that is not surfaceexposed and, accordingly, most likely not relevant in terms ofimmunogenicity/antigenicity.

Site-directed mutagenesis is another technique useful for elucidation ofa binding epitope. For example, in “alanine-scanning”, each residuewithin a protein segment is replaced with an alanine residue, and theconsequences for binding affinity measured. If the mutation leads to asignificant reduction in binding affinity, it is most likely involved inbinding. Monoclonal antibodies specific for structural epitopes (i.e.,antibodies which do not bind the unfolded protein) can be used to verifythat the alanine-replacement does not influence over-all fold of theprotein. See, e.g., Clackson and Wells, Science 1995; 267:383-386; andWells, Proc Natl Acad Sci USA 1996; 93:1-6.

Electron microscopy can also be used for epitope “foot-printing”. Forexample, Wang et al., Nature 1992; 355:275-278 used coordinatedapplication of cryoelectron micros-copy, three-dimensional imagereconstruction, and X-ray crystallography to determine the physicalfootprint of a Fab-fragment on the capsid surface of native cowpeamosaic virus.

Other forms of “label-free” assay for epitope evaluation include surfaceplasmon resonance (SPR, BIACORE) and reflectometric interferencespectroscopy (RifS). See, e.g., Fagerstam et al., Journal Of MolecularRecognition 1990; 3:208-14; Nice et al., J. Chroma-togr. 1993;646:159-168; Leipert et al., Angew. Chem. Int. Ed. 1998; 37:3308-3311;Kroger et al., Biosensors and Bioelectronics 2002; 17:937-944.

It should also be noted that an antibody binding the same orsubstantially the same epitope as an antibody of the invention can beidentified in one or more of the exemplary competition assays describedherein.

Upon immunization and production of antibodies in a vertebrate or cell,particular selection steps may be performed to isolate antibodies asclaimed. In this regard, in a specific embodiment, the invention alsorelates to methods of producing such antibodies, comprising: (a)immunizing a non-human mammal with an immunogen comprising a MICApolypeptide; and (b) preparing antibodies from said immunized animal;and (c) selecting antibodies from step (b) that are capable of bindingMICA.

Typically, an anti-MICA antibody provided by the invention has anaffinity for a MICA polypeptide in the range of about 10⁴ to about 10¹¹M⁻¹ (e.g., about 10⁸ to about 10¹⁰ M⁻¹). For example, in a particularaspect the invention provides Anti-MICA antibody that have an averagedisassociation constant (K_(D)) of less than 1×10⁻⁹ M with respect toMICA, as determined by, e.g., surface plasmon resonance (SPR) screening(such as by analysis with a BIAcore™ SPR analytical device). In a moreparticular exemplary aspect, the invention provides Anti-MICA antibodiesthat have a KD of about 1×10⁻⁸ M to about 1×10⁻¹⁰ M, or about 1×10⁻⁹ Mto about 1×10⁻¹¹ M, for MICA.

Antibodies can be characterized for example by a mean KD of no more thanabout (i.e. better affinity than) 100, 60, 10, 5, or 1 nanomolar,preferably sub-nanomolar or optionally no more than about 500, 200, 100or 10 picomolar. KD can be determined for example for example byimmobilizing recombinantly produced human MICA proteins on a chipsurface, followed by application of the antibody to be tested insolution. In one embodiment, the method further comprises a step (d),selecting antibodies from (b) that are capable of competing for bindingto MICA with antibody 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8,10F3, 15F9 or 14B4.

In one aspect of any of the embodiments, the antibodies preparedaccording to the present methods are monoclonal antibodies. In anotheraspect, the non-human animal used to produce antibodies according to themethods of the invention is a mammal, such as a rodent, bovine, porcine,fowl, horse, rabbit, goat, or sheep. The antibodies of the presentinvention encompass 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8,10F3, 15F9 or 14B4. Additionally, antibodies of the invention canoptionally be specified to be antibodies other than any of antibodiesBAMO1 or BAMO3 described in Salih et al. (2003) (Blood 102(4):1389-1396), antibody 2C10, 3H5, 6D4 or 6G6 described in Groh et al.(1996) Proc. Natl. Acad. Sci USA 93:12445-12450, Groh et al. (1998)Science 279:1737-1740 or WO2008/131406, the disclosures of each of whichare incorporated herein by reference, or derivatives of the foregoing,e.g. that comprise the CDRs or the antigen binding region in whole or inpart.

According to an alternate embodiment, the DNA encoding an antibody thatbinds an epitope present on MICA polypeptides is isolated from thehybridoma of this invention and placed in an appropriate expressionvector for transfection into an appropriate host. The host is then usedfor the recombinant production of the antibody, or variants thereof,such as a humanized version of that monoclonal antibody, activefragments of the antibody, chimeric antibodies comprising the antigenrecognition portion of the antibody, or versions comprising a detectablemoiety.

DNA encoding the monoclonal antibodies of the invention, e.g., antibody6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8, 10F3, 15F9 or 14B4, canbe readily isolated and sequenced using conventional procedures (e. g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of murine antibodies). Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. As describedelsewhere in the present specification, such DNA sequences can bemodified for any of a large number of purposes, e.g., for humanizingantibodies, producing fragments or derivatives, or for modifying thesequence of the antibody, e.g., in the antigen binding site in order tooptimize the binding specificity of the antibody. In one embodiment, theinvention comprises an isolated nucleic acid sequence encoding a lightchain and/or a heavy chain of an antibody (e.g. 6E4, 20C6, 16A8, 9C10,19E9, 12A10, 10A7, 18E8, 10F3, 15F9 or 14B4), as well as a recombinanthost cell comprising (e.g. in its genome) such nucleic acid.

Recombinant expression in bacteria of DNA encoding the antibody is wellknown in the art (see, for example, Skerra et al., Curr. Opinion inImmunol., 5, pp. 256 (1993); and Pluckthun, Immunol. 130, p. 151 (1992).

Assessing Activity

Once an antigen-binding compound is obtained it will generally beassessed for its ability to block an interaction between NKG2D and MICA(e.g. sMICA or membrane bound MICA), to block shedding of MICA from acell, to inhibit sMICA-induced downmodulation of NKG2D, to cause thedeath of a MICA-expressing cell, to induce ADCC or CDC towards, and/orto inhibit the proliferation of and/or cause the elimination ofMICA-expressing target cells.

Assessing the antigen-binding compound's ability to reduce binding orblock an interaction between MICA and NKG2D can be carried out at anysuitable stage of the method, e.g. as in the examples are providedherein. For example, tumor cells expressing MICA on their surface can bebrought into contact with cells (e.g. effector cells) expressing NKG2Don their surface, with or without the addition of a candidate anti-MICAantibody. Binding between the MICA- and NKG2D-expressing cells can beassessed, and an antibody that does not reduce binding is selected.Another possibility involves contacting an isolated MICA polypeptidewith an isolated NKG2D polypeptide, or a cell expressing an NKG2Dpolypeptide at its surface, and assessing binding between MICA and NKG2Dpolypeptide or cells expressing NKG2D. Another possibility involvescontacting an isolated NKG2D polypeptide with a cell expressing a MICApolypeptide at its surface, and assessing binding between MICApolypeptide or a cell expressing MICA.

For example, to determine whether an agent blocks MICA interactions withNKG2D, the following test is performed: The cell line C1R or RMAtransfected with MICA is incubated with a soluble NKG2D-Fc fusionprotein, in the presence or absence of increasing concentrations of atest anti-MICA mAb. The cells are washed, and then incubated with asecondary antibody that recognizes the Fc part of the NKG2D-Fc fusionprotein, washed again, and analyzed on a flow cytometer (FACScalibur,Beckton Dickinson), by standard methods. In the absence of anti-MICAmAbs, the NKG2D-Fc protein binds well to C1R or RMA cells. In thepresence of an anti-MICA mAb that blocks MICA binding to NKG2D, there isa reduction of binding of NKG2D-Fc to the cells.

Preferably, assessing the antigen-binding compound's ability to reducebinding or block an interaction between MICA and NKG2D can also becarried out by assessing the effect of the anti-MICA antibody on thefunction of NKG2D-expressing cells (e.g. NK or T cells). Preferably NKor T cells are used that express NKG2D but not CD16 so as to avoid anycontribution of a CD16-mediated ADCC effect. If an anti-MICA antibodyreduces or blocks MICA-NKG2D interactions it will be expected to dampenNKG2D-mediated activation of NK or T cells. An antibody that does notreduce binding or block an interaction between MICA and NKG2D willtherefore not substantially reduce or block NKG2D-mediated activation ofNK or T cells. This can be evaluated by a typical cytotoxicity assay,examples of which are described herein. Any of a number of cell-basedassays can be used to assess NKG2D activity, including geneexpression-based activities, cytotoxicity-based assays, andproliferation assays. In one aspect, in vitro assays will use NK cellsor T cells from human patients, or, e.g., T cell lines transfected withan NKG2D-encoding transgene, so long that the expression of the receptoralters the activity of the cells in a detectable way, e.g., renders themactivatable by NKG2D ligand. Any suitable physiological change thatreflects NKG2D activity can be used to assess the utility of a testcompound or antibody. For example, one can measure a variety of effects,such as changes in gene expression, cytokine production, cell growth,cell proliferation, pH, intracellular second messengers, e.g., Ca2+,IP3, cGMP, or cAMP, or activity such as cytotoxic activity or ability toactivate other T cells. In one embodiment, the activity of the receptoris assessed by detecting the expression of NKG2D-responsive genes, e.g.,CD25, IFN-gamma, or TNF-alpha (see, e.g., Groh et al. (2003) PNAS 100:9452-9457; André et al. (2004) Eur. J. Immunol 34: 1-11). In oneembodiment, NKG2D activity is assessed by incubating NKG2D+T or NK cellsin the presence of MICA-expressing cells and an anti-MICA antibody, andassessing the ability of the compound or test antibody to inhibit therelease of TNF-alpha or IFN-gamma by the T or NK cells.

Exemplary cytotoxicity assays are also described in the examples hereinwhere NKG2D-mediated killing of target cells is assessed. Here, theability of anti-MICA antibodies to reduce or inhibit the NKG2D+CD16−NK92 cell are used to assess NK cell-mediated killing ofMICA*019-transfected BaF/3 by measuring target cell release of 51Cr. Thein vitro cytotoxicity assay is carried out by standard methods that arewell known in the art, as described for example in Coligan et al., eds.,Current Protocols in Immunology, Greene Publishing Assoc. and WileyInterscience, N.Y., (1992, 1993). The MICA-expressing target cells arelabeled with ⁵¹Cr prior to addition of NK cells, and then the killing isestimated as proportional to the release of ⁵¹Cr from the cells to themedium, as a result of killing. Addition of an agent that reducesbinding or blocks an interaction between MICA and NKG2D results inprevention of the initiation and propagation of activatory signaling viaNKG2D. Therefore addition of such agents results in decreases inNK-mediated killing of the target cells.

An antigen-binding compound that does not reduce or block (e.g. noreduction, or a reduction of less than 5%, 10%, 20% or 30%) theactivation of cells by NKG2D (e.g. cytokine production, cell growth,cell proliferation, pH, intracellular second messengers, NK-mediatedkilling of MICA-expressing cells) is designated a “non-blocking” mAb. Anantigen-binding compound that reduces or blocks the activation of cellsby NKG2D is designated a “blocking” mAb.

Assessing the antigen-binding compound's ability to block shedding ofMICA from a MICA-expressing cell can be carried out at any suitablestage of the method, e.g. as in the examples are provided herein. In oneexample, a sample of cells is provided and soluble extracellular MICA isdetected using ELISA methods. In one example, an antigen-bindingcompound of the invention is administered to a mammal and the presenceor absence, or levels of, circulating sMICA is measured. Examples of invitro detection assays are described in Nolting et al. (2010) Virology406(1):12-20. Briefly, a commercially available MICA Elisa kit (Bamomab,Munich, Germany) can be used. Plates are coated overnight with thecapture anti-MICA mAb BAMO-1 at 2 μg/ml in PBS, then blocked by additionof 100 μl of 15% BSA for 2 h at 37° C. and washed. Standards and samplesare added and the plates and incubated for 2 h at 37° C. Plates arewashed and the detection mAb BAMO-3 at 5 μg/ml in 7.5% BSA-PBS was addedfor 2 h at 37° C. Plates were then washed and anti-mouse IgG2a-HRP(1:8000 in 7.5% BSA-PBS) is added for 1 h at 37° C. Plates are thenwashed and developed using the Tetramethylbenzidine Peroxidase SubstrateSystem (KPL, Gaithersburg, Md.). The absorbance is measured at 450 nm

Assessing the antigen-binding compound's ability to induce ADCC, CDC orotherwise (e.g. by delivery of a toxic agent) lead to the elimination orinhibition of activity of MICA-expressing target cells, can be carriedout at any suitable stage of the method, e.g. as in the examples areprovided herein. This assessment can be useful at one or more of thevarious steps involved in the identification, production and/ordevelopment of an antibody (or other compound) destined for therapeuticuse. For example, activity may be assessed in the context of a screeningmethod to identify candidate antigen-binding compounds, or in methodswhere an antigen-binding compound is selected and made human suitable(e.g. made chimeric or humanized in the case of an antibody), where acell expressing the antigen-binding compound (e.g. a host cellexpressing a recombinant antigen-binding compound) has been obtained andis assessed for its ability to produce functional antibodies (or othercompounds), and/or where a quantity of antigen-binding compound has beenproduced and is to be assessed for activity (e.g. to test batches orlots of product). Generally the antigen-binding compound will be knownto specifically bind to a MICA polypeptide. The step may involve testinga plurality (e.g., a very large number using high throughput screeningmethods or a smaller number) of antigen-binding compounds.

Testing CDC and ADCC can be carried out can be determined by variousassays including those described in the experimental examples herein.Testing ADCC typically involves assessing cell-mediated cytotoxicity inwhich a MICA-expressing target cell (e.g. a cancer or otherMICA-expressing cell) with bound anti-MICA antibody is recognized by aneffector cell (e.g. a leukocyte bearing Fc receptors), without theinvolvement of complement. A cell which does not express a MICA antigencan optionally be used as a control. Activation of NK cell cytotoxicityis assessed by measuring an increase in cytokine production (e.g. IFN-γproduction) or cytotoxicity markers (e.g. CD107 mobilization).Preferably the antibody of the invention will induce an increase incytokine production, expression of cytoxicity markers, or target celllysis of at least 20%, 50%, 80%, 100%, 200% or 500% in the presence oftarget (MICA-expressing) cells, compared to a control antibody (e.g. anantibody not binding to MICA, a MICA antibody having murine constantregions). In another example, lysis of target cells is detected, e.g. ina chromium release assay, preferably the antibody of the invention willinduce lysis of at least 10%, 20%, 30%, 40% or 50% of target cells.

Antibody CDR Sequences

Antibody 6E4

The amino acid sequence of the heavy chain variable region of antibody6E4 is listed as SEQ ID NO: 7, the amino acid sequence of the lightchain variable region is listed as SEQ ID NO: 8. The amino acidsequences of heavy and light chain variable region of antibody 6E4 fusedto a human chain constant region (heavy and light, respectively) arelisted as SEQ ID NOS: 9 and 10, respectively. In a specific embodiment,the invention provides an antibody that binds essentially the sameepitope or determinant as monoclonal antibodies 6E4; optionally theantibody comprises an antigen binding region of antibody 6E4. In any ofthe embodiments herein, antibody 6E4 can be characterized by its aminoacid sequence and/or nucleic acid sequence encoding it. In one preferredembodiment, the monoclonal antibody comprises the Fab or F(ab′)₂ portionof 6E4. Also provided is a monoclonal antibody that comprises the heavychain-variable region of 6E4. According to one embodiment, themonoclonal antibody comprises the three CDRs of the heavy chain variableregion of 6E4 Also provided is a monoclonal antibody that furthercomprises the variable light chain variable region of 6E4 or one, two orthree of the CDRs of the light chain variable region of 6E4. Optionallyany one or more of said light or heavy chain CDRs may contain one, two,three, four or five or more amino acid modifications (e.g.substitutions, insertions or deletions). Optionally, provided is anantibody where any of the light and/or heavy chain variable regionscomprising part or all of an antigen binding region of antibody 6E4 arefused to an immunoglobulin constant region of the human IgG type,optionally a human constant region, optionally a human IgG1 or IgG3isotype.

In another aspect, the invention provides a purified polypeptide whichencodes an antibody, wherein the antibody comprises: a HCDR1 regioncomprising an amino acid sequence SYYAMS, GFTFSY or GFTFSYYAMS as setforth in SEQ ID NOS: 11-13, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR2 regioncomprising an amino acid sequence TISRGGNYIYYTDSVKG or TISRGGNYIY as setforth in SEQ ID NOS: 14-15, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR3 regioncomprising an amino acid sequence ISDYDGAWLAY as set forth in SEQ ID NO:16, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous aminoacids thereof, wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region comprising anamino acid sequence RSSQSIIHTNGNTYLE as set forth in SEQ ID NO: 17, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; a LCDR2 region comprising an amino acid sequenceKISNRFS as set forth in SEQ ID NO: 18, or a sequence of at least 4, 5,6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one or more ofthese amino acids may be substituted by a different amino acid; a LCDR3region comprising an amino acid sequence FQGSHVPWT as set forth in SEQID NO: 19, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, wherein one or more of these amino acids may bedeleted or substituted by a different amino acid.

In another aspect, the invention provides an antibody that binds humanMICA, comprising:

(a) the heavy chain variable region of SEQ ID NO: 7, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(b) the light chain variable region of SEQ ID NO: 8, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(c) the heavy chain variable region of SEQ ID NO: 7, wherein one or moreof these amino acids may be substituted by a different amino acid; andthe light chain variable region of SEQ ID NO: 8, wherein one, two, threeor more amino acids may be substituted by a different amino acid; and/or

(d) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2) amino acid sequencesas shown in SEQ ID NO: 11 to 16, wherein one, two, three or more aminoacids in a CDR may be substituted by a different amino acid; and/or

(e) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 17, 18 and 19, wherein one, two, threeor more amino acids in a CDR may be substituted by a different aminoacid; and/or

(f) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 11 to 16, wherein one, two, three ormore amino acids in a CDR may be substituted by a different amino acid;and the light chain CDRs 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 17, 18 and 19, wherein one, two, threeor more amino acids in a CDR may be substituted by a different aminoacid; and/or

(g) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 7, wherein one, two, three or more amino acids ina CDR may be substituted by a different amino acid; and/or

(h) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 8, wherein one, two, three or more amino acids ina CDR may be substituted by a different amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains May be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forMICA binding with a monoclonal antibody of (a) to (h), above.

Antibody 20C6

The amino acid sequence of the heavy chain variable region of antibody20C6 is listed in SEQ ID NO: 20, the amino acid sequence of the lightchain variable region is listed as SEQ ID NO: 21. The amino acidsequences of the heavy and light chain variable regions of antibody 20C6fused to a heavy chain constant region (heavy and light, respectively,are listed as SEQ ID NOS: 22 and 23, respectively. In one embodiment,the invention provides an antibody that binds essentially the sameepitope or determinant as monoclonal antibody 20C6; optionally theantibody comprises an antigen binding region of antibody 20C6. In any ofthe embodiments herein, antibody 20C6 can be characterized by its aminoacid sequence and/or nucleic acid sequence encoding it. In one preferredembodiment, the monoclonal antibody comprises the Fab or F(ab′)₂ portionof 20C6. Also provided is a monoclonal antibody that comprises the heavychain variable region of 20C6. According to one embodiment, themonoclonal antibody comprises the three CDRs of the heavy chain variableregion of 20C6. Also provided is a monoclonal antibody that furthercomprises the variable light chain variable region of 20C6 or one, twoor three of the CDRs of the light chain variable region of 20C6.Optionally any one or more of said light or heavy chain CDRs may containone, two, three, four or five amino acid modifications (e.g.substitutions, insertions or deletions). Optionally, provided is anantibody where any of the light and/or heavy chain variable regionscomprising part or all of an antigen binding region of antibody 16A8 arefused to an immunoglobulin constant region of the IgG type, optionally ahuman constant region, optionally an IgG1 or IgG3 isotype.

In another aspect, the invention provides a purified polypeptide whichencodes an antibody, wherein the antibody comprises: a HCDR1 regioncomprising an amino acid sequence TSGMGVG, GFSLSTSG or GFSLSTSGMGVG asset forth in SEQ ID NOS: 24-26, or a sequence of at least 4, 5, 6, 7, 8,9 or 10 contiguous amino acids thereof, wherein one or more of theseamino acids may be substituted by a different amino acid; a HCDR2 regioncomprising an amino acid sequence HIWWDDDKYYNPSLK or HIWWDDDK as setforth in SEQ ID NOS: 27-28, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR3 regioncomprising an amino acid sequence RTQGYFDY as set forth in SEQ ID NO:29, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous aminoacids thereof, wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region comprising anamino acid sequence RASQSISDYLH as set forth in SEQ ID NO: 30, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; a LCDR2 region comprising an amino acid sequenceYASQSIS as set forth in SEQ ID NO: 31, or a sequence of at least 4, 5,6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one or more ofthese amino acids may be substituted by a different amino acid; and/or aLCDR3 region comprising an amino acid sequence QNGHSFPWT as set forth inSEQ ID NO: 32, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10contiguous amino acids thereof, wherein one or more of these amino acidsmay be deleted or substituted by a different amino acid, or where thesequence may comprise an insertion of one or more amino acids.

In another aspect, the invention provides an antibody that binds humanMICA, comprising:

(a) the heavy chain variable region of SEQ ID NO: 20, wherein one, two,three or more amino acids in a CDR may be substituted by a differentamino acid; and/or

(b) the light chain variable region of SEQ ID NO: 21, wherein one, two,three or more amino acids in a CDR may be substituted by a differentamino acid; and/or

(c) the heavy chain variable region of SEQ ID NO: 20, wherein one, two,three or more amino acids may be substituted by a different amino acid;and the light chain variable region of SEQ ID NO: 21, wherein one oramino acids may be substituted by a different amino acid; and/or

(d) the heavy chain. CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NO: 24-29, wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid;and/or

(e) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NO: 30, 31 and 32, respectively, whereinone, two, three or more amino acids in a CDR may be substituted by adifferent amino acid; and/or

(f) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NO: 24 to 29, wherein one, two, three ormore amino acids in a CDR may be substituted by a different amino acid;and the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NO: 30, 31 and 32, wherein one, two, threeor more amino acids in a CDR may be substituted by a different aminoacid; and/or

(g) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 20, wherein one, two, three or more amino acidsmay be substituted by a different amino acid; and/or

(h) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 21, wherein one, two, three or more amino acidsmay be substituted by a different amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forMICA binding with a monoclonal antibody of (a) to (h), above.

Antibody 16A8

The amino acid sequence of the heavy chain variable region of antibody16A8 is listed in SEQ ID NO: 33, the amino acid sequence of the lightchain variable region is listed as SEQ ID NO: 34. The amino acidsequences of the heavy and light chain variable regions of antibody 16A8fused to a heavy chain constant region (heavy and light, respectively,are listed as SEQ ID NOS: 35 and 36, respectively. In one embodiment,the invention provides an antibody that binds essentially the sameepitope or determinant as monoclonal antibodies 16A8; optionally theantibody comprises an antigen binding region of antibody 16A8. In any ofthe embodiments herein, antibody 16A8 can be characterized by its aminoacid sequence and/or nucleic acid sequence encoding it. In one preferredembodiment, the monoclonal antibody comprises the Fab or F(ab′)₂ portionof 16A8. Also provided is a monoclonal antibody that comprises the heavychain variable region of 16A8. According to one embodiment, themonoclonal antibody comprises the three CDRs of the heavy chain variableregion of 16A8. Also provided is a monoclonal antibody that furthercomprises the variable light chain variable region of 16A8 or one, twoor three of the CDRs of the light chain variable region of 16A8.Optionally any one or more of said light or heavy chain CDRs may containone, two, three, four or five amino acid modifications (e.g.substitutions, insertions or deletions). Optionally, provided is anantibody where any of the light and/or heavy chain variable regionscomprising part or all of an antigen binding region of antibody 16A8 arefused to an immunoglobulin constant region of the IgG type, optionally ahuman constant region, optionally an IgG1 or IgG3 isotype.

In another aspect, the invention provides a purified polypeptide whichencodes an antibody, wherein the antibody comprises: a HCDR1 regioncomprising an amino acid sequence RYAMS, GFTFSR or GFTFSRYAMS as setforth in SEQ ID NOS: 37-39, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR2 regioncomprising an amino acid sequence TIFSGGSYTYYPDSV or TIFSGGSY as setforth in SEQ ID NOS: 40-41, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR3 regioncomprising an amino acid sequence PNWERTFDY as set forth in SEQ ID NO:42, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous aminoacids thereof, wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region comprising anamino acid sequence KSSQSLLNSSNQKNYL as set forth in SEQ ID NO: 43, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; a LCDR2 region comprising an amino acid sequenceFASTRES as set forth in SEQ ID NO: 44, or a sequence of at least 4, 5,6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one or more ofthese amino acids may be substituted by a different amino acid; and/or aLCDR3 region comprising an amino acid sequence QQHYSTPPT as set forth inSEQ ID NO: 45, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10contiguous amino acids thereof, wherein one or more of these amino acidsmay be deleted or substituted by a different amino acid, or where thesequence may comprise an insertion of one or more amino acids.

In another aspect, the invention provides an antibody that binds humanMICA, comprising:

(a) the heavy chain variable region of SEQ ID NO: 33, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(b) the light chain variable region of SEQ ID NO: 34, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(c) the heavy chain variable region of SEQ ID NO: 33, wherein one, two,three or more amino acids may be substituted by a different amino acid;and the light chain variable region of SEQ ID NO: 34, wherein one ormore amino acids may be substituted by a different amino acid; and/or

(d) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 37-42, wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid;and/or

(e) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 43, 44 and 45, respectively, whereinone, two, three or more amino acids in a CDR may be substituted by adifferent amino acid; and/or

(f) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 37-42, wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid; andthe light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 43, 44 and 45, wherein one, two, threeor more amino acids in a CDR may be substituted by a different aminoacid; and/or

(g) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 33, wherein one, two, three or more acids may besubstituted by a different amino acid; and/or

(h) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 34, wherein one, two, three or more amino acidsmay be substituted by a different amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forMICA binding with a monoclonal antibody of (a) to (h), above.

Antibody 19E9

The amino acid sequence of the heavy chain variable region of antibody19E9 is listed as SEQ ID NO: 46, the amino acid sequence of the lightchain variable region is listed as SEQ ID NO: 47.

In a specific embodiment, the invention provides an antibody that bindsessentially the same epitope or determinant as monoclonal antibodies19E9; optionally the antibody comprises an antigen binding region ofantibody 19E9. In any of the embodiments herein, antibody 19E9 can becharacterized by its amino acid sequence and/or nucleic acid sequenceencoding it. In one preferred embodiment, the monoclonal antibodycomprises the Fab or F(ab′)₂ portion of 19E9. Also provided is amonoclonal antibody that comprises the heavy chain variable region of19E9. According to one embodiment, the monoclonal antibody comprises thethree CDRs of the heavy chain variable region of 19E9. Also provided isa monoclonal antibody that further comprises the variable light chainvariable region of 19E9 or one, two or three of the CDRs of the lightchain variable region of 19E9. Optionally any one or more of said lightor heavy chain CDRs may contain one, two, three, four or five or moreamino acid modifications (e.g. substitutions, insertions or deletions).

Optionally, provided is an antibody where any of the light and/or heavychain variable regions comprising part or all of an antigen bindingregion of antibody 19E9 are fused to an immunoglobulin constant regionof the human IgG type, optionally a human constant region, optionally ahuman IgG1 or IgG3 isotype.

In another aspect, the invention provides a purified polypeptide whichencodes an antibody, wherein the antibody comprises: a HCDR1 regioncomprising an amino acid sequence SDYAWN, GYSITSD or GYSITSDYAWN as setforth in SEQ ID NOS: 48-50, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR2 regioncomprising an amino acid sequence FVSYSGTTKYNPSLKS or FVSYSGTTK as setforth in SEQ ID NOS: 51-52, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR3 regioncomprising an amino acid sequence GYGFDY as set forth in SEQ ID NO: 53,or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; a LCDR1 region comprising an amino acid sequenceSATSSISSIYFH as set forth in SEQ ID NO: 54, or a sequence of at least 4,5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one or moreof these amino acids may be substituted by a different amino acid; aLCDR2 region comprising an amino acid sequence RTSNLAS as set forth inSEQ ID NO: 55, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10contiguous amino acids thereof, wherein one or more of these amino acidsmay be substituted by a different amino acid; a LCDR3 region comprisingan amino acid sequence QQGTTIPFT as set forth in SEQ ID NO: 56, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be deleted orsubstituted by a different amino acid.

In another aspect, the invention provides an antibody that binds humanMICA, comprising:

(a) the heavy chain variable region of SEQ ID NO: 46, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(b) the light chain variable region of SEQ ID NO: 47, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(c) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NO: 48-53, wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid;and/or

(d) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 54, 55 and 56, wherein one, two, threeor more amino acids in a CDR may be substituted by a different aminoacid; and/or

(e) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 48-53, wherein one or more amino acidsin a CDR may be substituted by a different amino acid; and the lightchain CDRs 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences asshown in SEQ ID NOS: 54, 55 and 56, wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid;and/or

(f) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 46, wherein one, two, three or more amino acidsmay be substituted by a different amino acid; and/or

(g) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 47, wherein one, two, three or more amino acidsmay be substituted by a different amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forMICA binding with a monoclonal antibody of (a) to (g), above.

Antibody 9C10

The amino acid sequence of the heavy chain variable region of antibody9C10 is listed as SEQ ID NO: 57, the amino acid sequence of the lightchain variable region is listed as SEQ ID NO: 58. In a specificembodiment, the invention provides an antibody that binds essentiallythe same epitope or determinant as monoclonal antibodies 9C10;optionally the antibody comprises an antigen binding region of antibody9C10. In any of the embodiments herein, antibody 9C10 can becharacterized by its amino acid sequence and/or nucleic acid sequenceencoding it. In one preferred embodiment, the monoclonal antibodycomprises the Fab or F(ab′)₂ portion of 9C10. Also provided is amonoclonal antibody that comprises the heavy chain variable region of9010. According to one embodiment, the monoclonal antibody comprises thethree CDRs of the heavy chain variable region of 9C10. Also provided isa monoclonal antibody that further comprises the variable light chainvariable region of 9C10 or one, two or three of the CDRs of the lightchain variable region of 9C10. Optionally any one or more of said lightor heavy chain CDRs may contain one, two, three, four or five or moreamino acid modifications (e.g. substitutions, insertions or deletions).Optionally, provided is an antibody where any of the light and/or heavychain variable regions comprising part or all of an antigen bindingregion of antibody 9C10 are fused to an immunoglobulin constant regionof the human IgG type, optionally a human constant region, optionally ahuman IgG1 or IgG3 isotype.

In another aspect, the invention provides a purified polypeptide whichencodes an antibody, wherein the antibody comprises: a HCDR1 regioncomprising an amino acid sequence RYWMN, GYSFTR or GYSFTRYWMN as setforth in SEQ ID NOS: 59-61, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR2 regioncomprising an amino acid sequence MIHPSDSETRLNQKFKD or MIHPSDSETR as setforth in SEQ ID NOS: 62-63, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR3 regioncomprising an amino acid sequence GNFFYVMDY as set forth in SEQ ID NO:64, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous aminoacids thereof, wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region comprising anamino acid sequence RASQSIGTSIH as set forth in SEQ ID NO: 65, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; a LCDR2 region comprising an amino acid sequenceASESISG as set forth in SEQ ID NO: 66; or a sequence of at least 4, 5,6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one or more ofthese amino acids may be substituted by a different amino acid; a LCDR3region comprising an amino acid sequence QQSNFWPFT as set forth in SEQID NO: 67, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, wherein one or more of these amino acids may bedeleted or substituted by a different amino acid.

In another aspect, the invention provides an antibody that binds humanMICA, comprising:

(a) the heavy chain variable region of SEQ ID NO: 67, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(b) the light chain variable region of SEQ ID NO: 68, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(c) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NO: 59-64, wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid;and/or

(d) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 65, 66 and 67, wherein one, two, threeor more amino acids in a CDR may be substituted by a different aminoacid; and/or

(e) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 59-64, wherein one or more amino acidsin a CDR may be substituted by a different amino acid; and the lightchain CDRs 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences asshown in SEQ ID NOS: 65, 66 and 67, wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid;and/or

(f) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 57, wherein one, two, three or more amino acidsmay be substituted by a different amino acid; and/or

(g) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 58, wherein one, two, three or more amino acidsmay be substituted by a different amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forMICA binding with a monoclonal antibody of (a) to (g), above.

Antibody 12A10

The amino acid sequence of the heavy chain variable region of antibody12A10 is listed as SEQ ID NO: 68, the amino acid sequence of the lightchain variable region is listed as SEQ ID NO: 69. In a specificembodiment, the invention provides an antibody that binds essentiallythe same epitope or determinant as monoclonal antibodies 12A10;optionally the antibody comprises an antigen binding region of antibody12A10. In any of the embodiments herein, antibody 12A10 can becharacterized by its amino acid sequence and/or nucleic acid sequenceencoding it. In one preferred embodiment, the monoclonal antibodycomprises the Fab or F(ab′)₂ portion of 12A10. Also provided is amonoclonal antibody that comprises the heavy chain variable region of12A10. According to one embodiment, the monoclonal antibody comprisesthe three CDRs of the heavy chain variable region of 12A10. Alsoprovided is a monoclonal antibody that further comprises the variablelight chain variable region of 12A10 or one, two or three of the CDRs ofthe light chain variable region of 12A10. Optionally any one or more ofsaid light or heavy chain CDRs may contain one, two, three, four or fiveor more amino acid modifications (e.g. substitutions, insertions ordeletions). Optionally, provided is an antibody where any of the lightand/or heavy chain variable regions comprising part or all of an antigenbinding region of antibody 12A10 are fused to an immunoglobulin constantregion of the human IgG type, optionally a human constant region,optionally a human IgG1 or IgG3 isotype.

In another aspect, the invention provides a purified polypeptide whichencodes an antibody, wherein the antibody comprises: a HCDR1 regioncomprising an amino acid sequence NYWMN, GYSFTN or GYSFTNYWMN as setforth in SEQ ID NOS: 70-72, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR2 regioncomprising an amino acid sequence MIHPSDSETRLNQKFKD or MIHPSDSETR as setforth in SEQ ID NOS: 73-74, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR3 regioncomprising an amino acid sequence DDFFTMDY as set forth in SEQ ID NO:75, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous aminoacids thereof, wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region comprising anamino acid sequence RASQNIVTSIH as set forth in SEQ ID NO: 76, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; a LCDR2 region comprising an amino acid sequenceYASESIS as set forth in SEQ ID NO: 77, or a sequence of at least 4, 5,6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one or more ofthese amino acids may be substituted by a different amino acid; a LCDR3region comprising an amino acid sequence QQSNIWPLT as set forth in SEQID NO: 78, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, wherein one or more of these amino acids may bedeleted or substituted by a different amino acid.

In another aspect, the invention provides an antibody that binds humanMICA, comprising:

(a) the heavy chain variable region of SEQ ID NO: 68, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(b) the light chain variable region of SEQ ID NO: 69, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(c) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NO: 70-75, wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid;and/or

(d) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 76, 77 and 78, wherein one, two, threeor more amino acids in a CDR may be substituted by a different aminoacid; and/or

(e) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 70-75, wherein one or more amino acidsin a CDR may be substituted by a different amino acid; and the lightchain CDRs 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences asshown in SEQ ID NOS: 76, 77 and 78, wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid;and/or

(f) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 68, wherein one, two, three or more amino acidsmay be substituted by a different amino acid; and/or

(g) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 69, wherein one, two, three or more amino acidsmay be substituted by a different amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forMICA binding with a monoclonal antibody of (a) to (g), above.

Antibody 10A7

The amino acid sequence of the heavy chain variable region of antibody10A7 is listed as SEQ ID NO: 79, the amino acid sequence of the lightchain variable region is listed as SEQ ID NO: 80. In a specificembodiment, the invention provides an antibody that binds essentiallythe same epitope or determinant as monoclonal antibodies 10A7;optionally the antibody comprises an antigen binding region of antibody10A7. In any of the embodiments herein, antibody 10A7 can becharacterized by its amino acid sequence and/or nucleic acid sequenceencoding it. In one preferred embodiment, the monoclonal antibodycomprises the Fab or F(ab′)₂ portion of 10A7. Also provided is amonoclonal antibody that comprises the heavy chain variable region of10A7. According to one embodiment, the monoclonal antibody comprises thethree CDRs of the heavy chain variable region of 10A7. Also provided isa monoclonal antibody that further comprises the variable light chainvariable region of 10A7 or one, two or three of the CDRs of the lightchain variable region of 10A7. Optionally any one or more of said lightor heavy chain CDRs may contain one, two, three, four or five or moreamino acid modifications (e.g. substitutions, insertions or deletions).Optionally, provided is an antibody where any of the light and/or heavychain variable regions comprising part or all of an antigen bindingregion of antibody 10A7 are fused to an immunoglobulin constant regionof the human IgG type, optionally a human constant region, optionally ahuman IgG1 or IgG3 isotype.

In another aspect, the invention provides a purified polypeptide whichencodes an antibody, wherein the antibody comprises: a HCDR1 regioncomprising an amino acid sequence TSGMGVG, GFSLSTSG or GFSLSTSGMGVG asset forth in SEQ ID NOS: 81-83, or a sequence of at least 4, 5, 6, 7, 8,9 or 10 contiguous amino acids thereof, wherein one or more of theseamino acids may be substituted by a different amino acid; a HCDR2 regioncomprising an amino acid sequence HIWWDDDRYYNPSLKS or HIWWDDDRY as setforth in SEQ ID. NOS: 84-85, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR3 regioncomprising an amino acid sequence RLNGYFDY as set forth in SEQ ID NO:86, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous aminoacids thereof, wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region comprising anamino acid sequence RASQSISDYLH as set forth in SEQ ID NO: 87, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; a LCDR2 region comprising an amino acid sequenceYASQSIS as set forth in SEQ ID NO: 88, or a sequence of at least 4, 5,6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one or more ofthese amino acids may be substituted by a different amino acid; a LCDR3region comprising an amino acid sequence QNGHSFPFT as set forth in SEQID NO: 89, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, wherein one or more of these amino acids may bedeleted or substituted by a different amino acid.

In another aspect, the invention provides an antibody that binds humanMICA, comprising:

(a) the heavy chain variable region of SEQ ID NO: 79, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(b) the light chain variable region of SEQ ID NO: 80, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(c) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NO: 81-86, wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid;and/or

(d) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 87, 88 and 89, wherein one, two, threeor more amino acids in a CDR may be substituted by a different aminoacid; and/or

(e) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 81 to 86, wherein one or more aminoacids in a CDR may be substituted by a different amino acid; and thelight chain CDRs 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequencesas shown in SEQ ID NOS: 87, 88 and 89, wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid;and/or

(f) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 79, wherein one, two, three or more amino acidsmay be substituted by a different amino acid; and/or

(g) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 80, wherein one, two, three or more amino acidsmay be substituted by a different amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forMICA binding with a monoclonal antibody of (a) to (g), above.

Antibody 18E8

The amino acid sequence of the heavy chain variable region of antibody18E8 is listed as SEQ ID NO: 90, the amino acid sequence of the lightchain variable region is listed as SEQ ID NO: 91. In a specificembodiment, the invention provides an antibody that binds essentiallythe same epitope or determinant as monoclonal antibodies 18E8;optionally the antibody comprises an antigen binding region of antibody18E8. In any of the embodiments herein, antibody 18E8 can becharacterized by its amino acid sequence and/or nucleic acid sequenceencoding it. In one preferred embodiment, the monoclonal antibodycomprises the Fab or F(ab′)₂ portion of 18E8. Also provided is amonoclonal antibody that comprises the heavy chain variable region of18E8. According to one embodiment, the monoclonal antibody comprises thethree CDRs of the heavy chain variable region of 18E8. Also provided isa monoclonal antibody that further comprises the variable light chainvariable region of 18E8 or one, two or three of the CDRs of the lightchain variable region of 18E8. Optionally any one or more of said lightor heavy chain CDRs may contain one, two, three, four or five or moreamino acid modifications (e.g. substitutions, insertions or deletions).Optionally, provided is an antibody where any of the light and/or heavychain variable regions comprising part or all of an antigen bindingregion of antibody 18E8 are fused to an immunoglobulin constant regionof the human IgG type, optionally a human constant region, optionally ahuman IgG1 or IgG3 isotype.

In another aspect, the invention provides a purified polypeptide whichencodes an antibody, wherein the antibody comprises: a HCDR1 regioncomprising an amino acid sequence SDYSWH, GYSITSD or GYSITSDYSWH as setforth in SEQ ID NOS: 92-94, or a sequence of at least 4, 6, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR2 regioncomprising an amino acid sequence NIHYSGRINYNPSLRS or NIHYSGRIN as setforth in SEQ ID NOS: 95-96, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR3 regioncomprising an amino acid sequence RRTFGNFEDY as set forth in SEQ ID NO:97, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous aminoacids thereof, wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region comprising anamino acid sequence RSSSSVNYMH as set forth in SEQ ID NO: 98, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; a LCDR2 region comprising an amino acid sequenceATSTLAS as set forth in SEQ ID NO: 99, or a sequence of at least 4, 5,6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one or more ofthese amino acids may be substituted by a different amino acid; a LCDR3region comprising an amino acid sequence QQWSSNPLT as set forth in SEQID NO: 100, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, wherein one or more of these amino acids may bedeleted or substituted by a different amino acid.

In another aspect, the invention provides an antibody that binds humanMICA, comprising:

(a) the heavy chain variable region of SEQ ID NO: 90, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(b) the light chain variable region of SEQ ID NO: 91, wherein one, two,three or

(c) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NO: 92-97, wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid;and/or

(d) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 98, 99 and 100, wherein one, two,three or more amino acids in a CDR may be substituted by a differentamino acid; and/or

(e) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 92-97, wherein one or more amino acidsin a CDR may be substituted by a different amino acid; and the lightchain CDRs 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences asshown in SEQ ID NOS: 98, 99 and 100, wherein one, two, three or moreamino acids in a CDR may be substituted by a different amino acid;and/or

(f) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 90, wherein one, two, three or more amino acidsmay be substituted by a different amino acid; and/or

(g) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 91, wherein one, two, three or more amino acidsmay be substituted by a different amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forMICA binding with a monoclonal antibody of (a) to (g), above.

Antibody 10F3

The amino acid sequence of the heavy chain variable region of antibody10F3 is listed as SEQ ID NO: 101, the amino acid sequence of the lightchain variable region is listed as SEQ ID NO: 102. In a specificembodiment, the invention provides an antibody that binds essentiallythe same epitope or determinant as monoclonal antibodies 10F3;optionally the antibody comprises an antigen binding region of antibody10F3. In any of the embodiments herein, antibody 10F3 can becharacterized by its amino acid sequence and/or nucleic acid sequenceencoding it. In one preferred embodiment, the monoclonal antibodycomprises the Fab or F(ab′)₂ portion of 10F3. Also provided is amonoclonal antibody that comprises the heavy chain variable region of10F3. According to one embodiment, the monoclonal antibody comprises thethree CDRs of the heavy chain variable region of 10F3. Also provided isa monoclonal antibody that further comprises the variable light chainvariable region of 10F3 or one, two or three of the CDRs of the lightchain variable region of 10F3. Optionally any one or more of said lightor heavy chain CDRs may contain one, two, three, four or five or moreamino acid modifications (e.g. substitutions, insertions or deletions).Optionally, provided is an antibody where any of the light and/or heavychain variable regions comprising part or all of an antigen bindingregion of antibody 10F3 are fused to an immunoglobulin constant regionof the human IgG type, optionally a human constant region, optionally ahuman IgG1 or IgG3 isotype.

In another aspect, the invention provides a purified polypeptide whichencodes an antibody, wherein the antibody comprises: a HCDR1 regioncomprising an amino acid sequence SYTMH, GYTFTS or GYTFTSYTMH as setforth in SEQ ID NOS: 103-105, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR2 regioncomprising an amino acid sequence YINPSSGYTEYNQKFKD or YINPSSGYTE as setforth in SEQ ID NOS: 106-107, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR3 regioncomprising an amino acid sequence GGDWDVDWFVY as set forth in SEQ ID NO:108, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous aminoacids thereof, wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region comprising anamino acid sequence SASSSISYMH as set forth in SEQ ID NO: 109, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; a LCDR2 region comprising an amino acid sequenceSTSKLAS as set forth in SEQ ID NO: 110, or a sequence of at least 4, 5,6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one or more ofthese amino acids may be substituted by a different amino acid; a LCDR3region comprising an amino acid sequence QHRSTYPFT as set forth in SEQID NO: 111, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, wherein one or more of these amino acids may bedeleted or substituted by a different amino acid.

In another aspect, the invention provides an antibody that binds humanMICA, comprising:

(a) the heavy chain variable region of SEQ ID NO: 101, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(b) the light chain variable region of SEQ ID NO: 102, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(c) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NO: 103-108, wherein one, two, three ormore amino acids in a CDR may be substituted by a different amino acid;and/or

(d) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 109, 110 and 111, wherein one, two,three or more amino acids in a CDR may be substituted by a differentamino acid; and/or

(e) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 103-108, wherein one or more aminoacids in a CDR may be substituted by a different amino acid; and thelight chain CDRs 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequencesas shown in SEQ ID NOS: 109, 110 and 111, wherein one, two, three ormore amino acids in a CDR may be substituted by a different amino acid;and/or

(f) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 101, wherein one, two, three or more amino acidsmay be substituted by a different amino acid; and/or

(g) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 102, wherein one, two, three or more amino acidsmay be substituted by a different amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forMICA binding with a monoclonal antibody of (a) to (g), above.

Antibody 15F9

The amino acid sequence of the heavy chain variable region of antibody15F9 is listed as SEQ ID NO: 112, the amino acid sequence of the lightchain variable region is listed as SEQ ID NO: 113. In a specificembodiment, the invention provides an antibody that binds essentiallythe same epitope or determinant as monoclonal antibodies 15F9;optionally the antibody comprises an antigen binding region of antibody15F9. In any of the embodiments herein, antibody 15F9 can becharacterized by its amino acid sequence and/or nucleic acid sequenceencoding it. In one preferred embodiment, the monoclonal antibodycomprises the Fab or F(ab′)₂ portion of 15F9. Also provided is amonoclonal antibody that comprises the heavy chain variable region of15F9. According to one embodiment, the monoclonal antibody comprises thethree CDRs of the heavy chain variable region of 15F9. Also provided isa monoclonal antibody that further comprises the variable light chainvariable region of 15F9 or one, two or three of the CDRs of the lightchain variable region of 15F9. Optionally any one or more of said lightor heavy chain CDRs may contain one, two, three, four or five or moreamino acid modifications (e.g. substitutions, insertions or deletions).Optionally, provided is an antibody where any of the light and/or heavychain variable regions comprising part or all of an antigen bindingregion of antibody 15F9 are fused to an immunoglobulin constant regionof the human IgG type, optionally a human constant region, optionally ahuman IgG1 or IgG3 isotype.

In another aspect, the invention provides a purified polypeptide whichencodes an antibody, wherein the antibody comprises: a HCDR1 regioncomprising an amino acid sequence SGYSWH, GYSITSG or GYSITSGYSWH as setforth in SEQ ID NOS: 114-116, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR2 regioncomprising an amino acid sequence FIHYSGSTDYNPSLKS or FIHYSGSTD as setforth in SEQ ID NOS: 117-118, or a sequence of at least 4, 5, 6, 7, 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR3 regioncomprising an amino acid sequence DYGHWYFDV as set forth in SEQ ID NO:119, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous aminoacids thereof, wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region comprising anamino acid sequence KASQSVSYDVA as set forth in SEQ ID NO: 120, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; a LCDR2 region comprising an amino acid sequenceYASNRYT as set forth in SEQ ID NO: 121, or a sequence of at least 4, 5,6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one or more ofthese amino acids may be substituted by a different amino acid; a LCDR3region comprising an amino acid sequence QQDYSSLT as set forth in SEQ IDNO: 122, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, wherein one or more of these amino acids may bedeleted or substituted by a different amino acid.

In another aspect, the invention provides an antibody that binds humanMICA, comprising:

(a) the heavy chain variable region of SEQ ID NO:112, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(b) the light chain variable region of SEQ ID NO: 113, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(c) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NO: 114-119, wherein one, two, three ormore amino acids in a CDR may be substituted by a different amino acid;and/or

(d) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 120, 121 and 122, wherein one, two,three or more amino acids in a CDR may be substituted by a differentamino acid; and/or

(e) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 114-119, wherein one or more aminoacids in a CDR may be substituted by a different amino acid; and thelight chain CDRs 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequencesas shown in SEQ ID NOS: 120, 121 and 122, wherein one, two, three ormore amino acids in a CDR may be substituted by a different amino acid;and/or

(f) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 112, wherein one, two, three or more amino acidsmay be substituted by a different amino acid; and/or

(g) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 113, wherein one, two, three or more amino acidsmay be substituted by a different amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forMICA binding with a monoclonal antibody of (a) to (g), above.

Antibody 14B4

The amino acid sequence of the heavy chain variable region of antibody14B4 is listed as SEQ ID NO: 123, the amino acid sequence of the lightchain variable region is listed as SEQ ID NO: 124. In a specificembodiment, the invention provides an antibody that binds essentiallythe same epitope or determinant as monoclonal antibodies 14B4;optionally the antibody comprises an antigen binding region of antibody14B4. In any of the embodiments herein, antibody 14B4 can becharacterized by its amino acid sequence and/or nucleic acid sequenceencoding it. In one preferred embodiment, the monoclonal antibodycomprises the Fab or F(ab′)₂ portion of 14B4. Also provided is amonoclonal antibody that comprises the heavy chain variable region of14B4. According to one embodiment, the monoclonal antibody comprises thethree CDRs of the heavy chain variable region of 14B4. Also provided isa monoclonal antibody that further comprises the variable light chainvariable region of 14B4 or one, two or three of the CDRs of the lightchain variable region of 14B4. Optionally any one or more of said lightor heavy chain CDRs may contain one, two, three, four or five or moreamino acid modifications (e.g. substitutions, insertions or deletions).Optionally, provided is an antibody where any of the light and/or heavychain variable regions comprising part or all of an antigen bindingregion of antibody 14B4 are fused to an immunoglobulin constant regionof the human IgG type, optionally a human constant region, optionally ahuman IgG1 or IgG3 isotype.

In another aspect, the invention provides a purified polypeptide whichencodes an antibody, wherein the antibody comprises: a HCDR1 regioncomprising an amino acid sequence SYWMN, GYSFTS or GYSFTSYWMN, or G asset forth in SEQ ID NOS: 125-127, or a sequence of at least 4, 5, 6, 7,8, 9 or 10 contiguous amino acids thereof, wherein one or more of theseamino acids may be substituted by a different amino acid; a HCDR2 regioncomprising an amino acid sequence MIHPSDSETRLNQKFKD or MIHPSDSETR as setforth in SEQ ID NOS: 128-129, or a sequence of at least 4, 5, 6, 7; 8, 9or 10 contiguous amino acids thereof, wherein one or more of these aminoacids may be substituted by a different amino acid; a HCDR3 regioncomprising an amino acid sequence EMGPYTLDY as set forth in SEQ ID NO:130, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous aminoacids thereof, wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region comprising anamino acid sequence RASQNIDTSIH as set forth in SEQ ID NO: 131, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, wherein one or more of these amino acids may be substituted bya different amino acid; a LCDR2 region comprising an amino acid sequenceYASESIS as set forth in SEQ ID NO: 132, or a sequence of at least 4, 5,6, 7, 8, 9 or 10 contiguous amino acids thereof, wherein one or more ofthese amino acids may be substituted by a different amino acid; a LCDR3region comprising an amino acid sequence QQSNYWPLT as et forth in SEQ IDNO: 133, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, wherein one or more of these amino acids may bedeleted or substituted by a different amino acid.

In another aspect, the invention provides an antibody that binds humanMICA, comprising:

(a) the heavy chain variable region of SEQ ID NO: 123, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(b) the light chain variable region of SEQ ID NO: 124, wherein one, two,three or more amino acids may be substituted by a different amino acid;and/or

(c) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NO: 125-130, wherein one, two, three ormore amino acids in a CDR may be substituted by a different amino acid;and/or

(d) the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acidsequences as shown in SEQ ID NOS: 131, 132 and 133, wherein one, two,three or more amino acids in a CDR may be substituted by a differentamino acid; and/or

(e) the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acidsequences as shown in SEQ ID NOS: 125-130, wherein one or more aminoacids in a CDR may be substituted by a different amino acid; and thelight chain CDRs 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequencesas shown in SEQ ID NOS: 131, 132 and 133, wherein one, two, three ormore amino acids in a CDR may be substituted by a different amino acid;and/or

(f) the heavy chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 123, wherein one, two, three or more amino acidsmay be substituted by a different amino acid; and/or

(g) the light chain variable region which is at least 60%, 70%, 80%,85%, 90% or 95% identical to the variable region having an amino acidsequence of SEQ ID NO: 124, wherein one, two, three or more amino acidsmay be substituted by a different amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized by a sequenceof at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof,and/or as having an amino acid sequence that shares at least 50%, 60%,70%, 80%, 85%, 90% or 95% sequence identity with the particular CDR orset of CDRs listed in the corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forMICA binding with a monoclonal antibody of (a) to (g), above.

In any of the antibodies of the invention, e.g., 6E4, 20C6, 16A8, 9C10,19E9, 12A10, 10A7, 18E8, 10F3, 15F9 or 14B4, the specified variableregion and CDR sequences may comprise conservative sequencemodifications (1, 2, 3, 4, 5, 6, 7, 8 or more sequence modifications). Aconservative sequence modification refers to an amino acid modificationthat does not significantly affect or alter the binding characteristicsof the antibody containing the amino acid, sequence. Such conservativemodifications include amino acid substitutions, additions and deletions.Modifications can be introduced into an antibody of the invention bystandard techniques known in the art, such as site-directed mutagenesisand PCR-mediated mutagenesis. Conservative amino acid substitutions aretypically those in which an amino acid residue is replaced with an aminoacid residue having a side chain with similar physicochemicalproperties. Specified variable region and CDR sequences may compriseone, two, three, four or more amino acid insertions, deletions orsubstitutions. Where substitutions are made, preferred substitutionswill be conservative modifications. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g. glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g. threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Thus, one or more amino acid residues within theCDR regions of an antibody of the invention can be replaced with otheramino acid residues from the same side chain family and the alteredantibody can be tested for retained function (i.e., the properties setforth herein) using the assays described herein.

The term “identity” or “identical”, when used in a relationship betweenthe sequences of two or more polypeptides, refers to the degree ofsequence relatedness between polypeptides, as determined by the numberof matches between strings of two or more amino acid residues.“Identity” measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., “algorithms”).Identity of related polypeptides can be readily calculated by knownmethods. Such methods include, but are not limited to, those describedin Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

Preferred methods for determining identity are designed to give thelargest match between the sequences tested. Methods of determiningidentity are described in publicly available computer programs.Preferred computer program methods for determining identity between twosequences include the GCG program package, including GAP (Devereux etal., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group,University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA(Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX programis publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well knownSmith Waterman algorithm may also be used to determine identity.

The sequences of the CDRs, according to AbM (Oxford Molecular's AbMantibody modelling software definition), Kabat and Chothia definitionssystems, have been summarized in Table A below. While any suitablenumbering system may be used to designated CDR regions, in the absenceof any other indication, the numbering used herein is Abm. Suchnumbering has been established using the following indications: CDR-L1:Start: approx residue 24, residue before: always a Cys, residue after:always a Trp (typically Trp-Tyr-Gln, but also, Trp-Leu-Gln, Trp-Phe-Gln,Trp-Tyr-Leu), length: 10 to 17 residues; CDR-L2: Start: always 16residues after the end of L1, Residues before: generally Ile-Tyr (butalso, Val-Tyr, Ile-Lys, Ile-Phe), Length: always 7 residues; CDR-L3,Start: always 33 residues after end of L2, Residue before: always Cys,Residues after: always Phe-Gly-Xaa-Gly, Length: 7 to 11 residues;CDR-H1, Start: approx residue 26 (always 4 after a Cys) (Chothia/AbMdefinition, the Kabat definition starts 5 residues later), Residuesbefore: always Cys-Xaa-Xaa-Xaa, Residues after: always a Trp (typicallyTrp-Val, but also, Trp-Ile, Trp-Ala), Length: 10 to 12 residues (AbMdefinition, Chothia definition excludes the last 4 residues); CDR-H2,Start: always 15 residues after the end of Kabat/AbM definition ofCDR-H1, Residues before: typically Leu-Glu-Trp-Ile-Gly (but a number ofvariations, Residues afterLys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala), Length: Kabatdefinition 16 to 19 residues; AbM (and Chothia) definition ends 7residues earlier; CDR-H3, Start: always 33 residues after end of CDR-H2(always 2 after a Cys), Residues before: always Cys-Xaa-Xaa (typicallyCys-Ala-Arg), Residues after: always Trp-Gly-Xaa-Gly, Length: 3 to 25residues.

The sequences of the variable chains of the antibodies according to theinvention are listed in Table B below, with the leader sequenceunderlined at the beginning of each sequence (any antibody chain can bespecified to start at the amino acid position immediately following theend of the leader sequence), and each CDRs underlined. In any embodimentherein, a VL or VH sequence can be specified or numbered so as tocontain or lack a signal peptide or any part thereof.

In one embodiment, the antibodies of the invention are of the human IgG1or IgG3 isotype. In one embodiment, the antibodies of the invention areantibody fragments that retain their binding and/or functionalproperties.

TABLE A HCDR1 HCDR2 HCDR3 CDR SEQ SEQ SEQ mAb definition ID Sequence IDSequence ID Sequence 6E4 Kabat  11 SYYAMS  14 TISRGGNYIYYTDSVKG  16ISDYDGAWLAY Chotia  12 GFTFSY  15 TISRGGNYIY ISDYDGAWLAY Abm  13GFTFSYYAMS TISRGGNYIY ISDYDGAWLAY 20C6  Kabat  24 TSGMGVG  27HIWWDDDKYYNPSLK  29 RTQGYFDY Chotia  25 GFSLSTSG  28 HIWWDDDK RTQGYFDYAbm  26 GFSLSTSGMGVG HIWWDDDK RTQGYFDY 16A8  Kabat  37 RYAMS  40TIFSGGSYTYYPDSV  42 PNWERTFDY Chotia  38 GFTFSR  41 TIFSGGSY PNWERTFDYAbm  39 GFTFSRYAMS TIFSGGSY PNWERTFDY 19E9  Kabat  48 SDYAWN  51FVSYSGTTKYNPSLKS  53 GYGFDY Chotia  49 GYSITSD  52 FVSYSGTTK GYGFDY Abm 50 GYSITSDYAWN FVSYSGTTK GYGFDY  9C10 Kabat  59 RYWMN  62MIHPSDSETRLNQKFKD  64 GNFFYVMDY Chotia  60 GYSFTR  63 MIHPSDSETRGNFFYVMDY Abm  61 GYSFTRYWMN MIHPSDSETR GNFFYVMDY 12A10 Kabat  70 NYWMN 73 MIHPSDSETRLNQKFKD  75 DDFFTMDY Chotia  71 GYSFTN  74 MIHPSDSETRDDFFTMDY Abm  72 GYSFTNYWMN MIHPSDSETR DDFFTMDY 10A7  Kabat  81 TSGMGVG 84 HIWWDDDRYYNPSLKS  86 RLNGYFDY Chotia  82 GFSLSTSG  85 HIWWDDDRYRLNGYFDY Abm  83 GFSLSTSGMGVG HIWWDDDRY RLNGYFDY 18E8  Kabat  92 SDYSWH 95 NIHYSGRINYNPSLRS  97 RRTFGNFEDY Chotia  93 GYSITSD  96 NIHYSGRINRRTFGNFEDY Abm  94 GYSITSDYSWH NIHYSGRIN RRTFGNFEDY 10F3  Kabat 103SYTMH 106 YINPSSGYTEYNQKFKD 108 GGDWDVDWFVY Chotia 104 GYTFTS 107YINPSSGYTE GGDWDVDWFVY Abm 105 GYTFTSYTMH YINPSSGYTE GGDWDVDWFVY 15F9 Kabat 114 SGYSWH 117 FIHYSGSTDYNPSLKS 119 DYGHWYFDV Chotia 115 GYSITSG118 FIHYSGSTD DYGHWYFDV Abm 116 GYSITSGYSWH FIHYSGSTD DYGHWYFDV 14B4 Kabat 125 SYWMN 128 MIHPSDSETRLNQKFKD 130 EMGPYTLDY Chotia 126 GYSFTS129 MIHPSDSETR EMGPYTLDY Abm 127 GYSFTSYWMN MIHPSDSETR EMGPYTLDY LCDR1LCDR2 LCDR3 CDR SEQ SEQ SEQ mAb definition ID Sequence ID Sequence IDSequence 6E4 Kabat  17 RSSQSIIHTNGNTYLE  18 KISNRFS  19 FQGSHVPWT 20C6 Kabat  30 RASQSISDYLH  31 YASQSIS  32 QNGHSFPWT 16A8  Kabat  43KSSQSLLNSSNQKNYL  44 FASTRES  45 QQHYSTPPT 19E9   54 SATSSISSIYFH  55RTSNLAS  56 QQGTTIPFT  9C10  65 RASQSIGTSIH  66 ASESISG  67 QQSNFWPFT12A10 Kabat,  76 RASQNIVTSIH  77 YASESIS  78 QQSNIWPLT Chotia, Abm 10A7 Kabat,  87 RASQSISDYLH  88 YASQSIS  89 QNGHSFPFT Chotia, Abm 18E8 Kabat,  98 RSSSSVNYMH  99 ATSTLAS 100 QQWSSNPLT Chotia, Abm 10F3  Kabat,109 SASSSISYMH 110 STSKLAS 111 QHRSTYPFT Chotia, Abm 15F9  Kabat, 120KASQSVSYDVA 121 YASNRYT 122 QQDYSSLT Chotia, Abm 14B4  Kabat, 131RASQNIDTSIH 132 YASESIS 133 QQSNYWPLT Chotia, Abm

TABLE B Antibody SEQ portion ID NO Sequence   6E4 VH   7M N F V L S L I F L A L I L K G V Q C E V Q L V E SG G A L V K P G G S L K L S C A A S G F T F S Y Y AM S W V R Q T P E K R L E W V A T I S R G G N Y I YY T D S V K G R F T I S R D N A K N T L Y L Q M T SL R S E D T A M F Y C A S I S D Y D G A W L A Y W G Q G T L V T V  6E4 VL   8 M K L P V R L L V L M F W I P V S S S D V L M T Q TP L S L P V S L G D Q A S I S C R S S Q S I I H T NG N T Y L E W Y L Q K P G Q S P K L L I Y K I S N RF S G V P D R F S G S G S G T D F T L K I S R V E AE D L G V Y Y C F Q G S H V P W T F G G G T K L E I K  20C6 VH  20M D R L T S S F L L L I V P A Y V L S Q I T L K E SG P G I L K P S Q T L S L T C S F S G F S L S T S GM G V G W I R Q P S G K G L E W L A H I W W D D D KY Y N P S L K S Q L T I S K D T S R N Q V F L R I TS V D T A D T A T Y Y C A R R T Q G Y F D Y W G Q G T T L T V S S 20C6 VL  21 M V S T S Q L L G L L L F W T S A S R C D I V N T QS P A T L S V T P G D R V S L S C R A S Q S I S D YL H W Y Q Q K S H E S P R L L I K Y A S Q S I S G IP S R F S G S G S G S D F T L S I N S V E P E D V GV Y Y C Q N G H S F P W T F G G G T K L E I K  16A8 VH  33M N F V L S L I F L A L I L K G V R C E V Q L V E SG G G L V K P G G S L K L S C A A S G F T F S R Y AM S W V R Q T P E K R L E W V A T I F S G G S Y T YY P D S V K G R F T I S R D N A N N T L Y L Q M S SL K A E D T A M Y F C A R P N W E R T F D Y W G Q G T T L T V S S 16A8 VL  34 M E S Q T Q V L M F L L L W V S G A C T D I V M T QS P S S L A M S V G Q K V T M S C K S S Q S L L N SS N Q K N Y L A W Y Q Q K P G Q S P K L L V Y F A ST R E S G V P D R F M G S G S G T D F T L T I S S VQ A E D L A D Y F C Q Q H Y S T P P T F G G G T K L E I K  19E9 VH  46M R V L I L L W L F T A F P G L L S D V Q L Q E S GP G L V K P S Q S L S L T C T V T G Y S I T S D Y AW N W I R Q F P G N K L E W M G F V S Y S G T T K YN P S L K S R I S I T R D T S E N Q F F L Q L N S VT S E D T A T Y Y C A R G Y G F D Y W G Q G T T L T V S S  19E9 VL  47M Q I I S L L L I S V T V I V S N G E I V L T Q S PT T M A A S P G E K I T I T C S A T S S I S S I Y FH W Y Q Q R P G F S P K L L I Y R T S N L A S G V PA R F S G S G S G T S Y S L T I G T M E A E D V A TY Y C Q Q G T T I P F T F G S G T K L E I K  9C10 VH  57M G W S S I I L F L V A T S T G V H S Q V Q L Q Q PG A E L V R P G T S V N L S C K A S G Y S F T R Y WM N W V K Q R P G Q G L E W I G M I H P S D S E T RL N Q K F K D K A T L T V D K S S S T A Y M Q L S SP T S E D S A V Y Y C G Y G N F F Y V M D Y W G Q G T S V T V S S 9C10 VL  58 M V S T P Q F L V F L L F W I P A S R G D I L L T QS P A I L S V S P G E R V S F S C R A S Q S I G T SI H W Y Q Q R T N G S P R L L I K F A S E S I S G IP S R F S G S G S G T D F T L N I N S V E S E D I AD Y Y C Q Q S N F W P F T F G S G T K L E V K 12A10 VH  68M E W S W V F L F F L S V T T G V H S Q V Q L Q Q SG A D L V R P G A S V R L S C R A S G Y S F T N Y WM N W V K Q R P G Q G L E W I G M I H P S D S E T RL N Q K F K D K A T L T V D K S S N T A Y M Q L S SP T S E D S A I Y Y C A R D D F F T M D Y W G Q G T S V T V S S A S T K12A10 VL  69 M S V P T Q V L G L L L L W L T D A R C D I L L T QS P A I L S V S P G E R V S F S C R A S Q N I V T SI H W Y Q Q S T N G S P R L L I K Y A S E S I S G IP S R F S G S G S G T D F T L T I N S V E S E D V AD Y Y C Q Q S N I W P L T F G A G T K L E L K  10A7 VH  79M E W S W V F L F F L S V T T G V H S Q V T L K E SG P G I L K P S Q T L S L T C S F S G F S L S T S GM G V G W I R Q P S G K G L E W L A H I W W D D D RY Y N P S L K S Q L T I S K D T S R N Q V F L K I TS V D T A D T A T Y Y C A R R L N G Y F D Y W G Q GT T L T V S S A S T K  10A7 VL  80M S V P T Q V L G L L L L W L T D A R C D I V N T QS P A T L S V T L G D R V S L S C R A S Q S I S D YL H W Y Q Q K S H E S P R L L I K Y A S Q S I S G IP S R F S G S G S G S D F T L S I N S V E P E D V GV Y Y C Q N G H S F P F T F G S G T K L E I K  18E8 VH  90M E W S W V F L F F L S V T T G V H S D V Q L Q E SG P D L V N P S Q S L S L I C T V T G Y S I T S D YS W H W I R Q F P G N K L E W M G N I H Y S G R I NY N P S L R S R I S I T R D T S K N Q F F L Q L I SV T T E D T A T Y Y C A T R R T F G N F E D Y W G QG T T L T V S S A S T K  18E8 VL  91M S V P T Q V L G L L L L W L T D A R C Q I V L S QS P A T L S V S P G E K V T M T C R S S S S V N Y MH W Y Q Q K P G S S P K P W I Y A T S T L A S G V PA R F S G S G S G T S Y S L T I S R V E A E D A A TY Y C Q Q W S S N P L T F G A G T K L E L K  10F3 VH 101M E W S W V F L F F L S V T T G V H S Q V Q L Q Q SA A E L A R P G A S V K M S C K A S G Y T F T S Y TM H W V K Q R P G Q G L E W I G Y I N P S S G Y T EY N Q K F K D K T T L T V D K S S T T S Y M Q L S SL T S D N S A V Y Y C A R G G D W D V D W F V Y W GQ G T L V T V S A A S T K  10F3 VL 102M S V P T Q V L G L L L L W L T D A R C Q I V L T QS P A I M S A S P G E K V T I T C S A S S S I S Y MH W F Q Q K P G T S P K L W I Y S T S K L A S G V PA R F S G S G S G T S H S L T I S R M E A E D A A TY Y C Q H R S T Y P F T F G S G T K L E I K  15F9 VH 112M E W S W V F L F F L S V T T G V H S D V Q L Q E SG P D L V K P S Q S L S L T C T V T G Y S I T S G YS W H W I R Q F P G N K L E W M G F I H Y S G S T DY N P S L K S R I S L T R D T S K N Q F F L Q L N SV S T E D T A T Y Y C A K D Y G H W Y F D V W G A GT T V T V S S A S T K  15F9 VL 113M S V P T Q V L G L L L L W L T D A R C S I V M T QT P K F L L V S A G D R V T I T C K A S Q S V S Y DV A W Y Q Q K P G Q S P K L L I F Y A S N R Y T G VP A R F T G S G Y G T D F T F T I S T V Q A E D L AV Y F C Q Q D Y S S L T F G A G T K L E L K  14B4 VH 123M E W S W V F L F F L S V T T G V H S Q V Q L Q Q PG A E L V R P G A S V K L S C K A S G Y S F T S Y WM N W M K Q R P G Q G L E W I G M I H P S D S E T RL N Q K F K D K A T L T V D K S S S T A Y M Q L N SP T S E D S A V Y Y C A R E M G P Y T L D Y W G Q GT S V T V S S A S T K  14B4 VL 124M S V P T Q V L G L L L L W L T D A R C D I L L T QS P A I L S V S P G A R V S F S C R A S Q N I D T SI H W Y Q Q R T N G S P R L L I K Y A S E S I S G IP S R F S G S G S G T D F T L S I N S V E S E D I AD Y Y C Q Q S N Y W P L T F G A G T K L E L K

In one aspect of any of the embodiments of the invention, any antibodyof the invention may comprise a heavy and/or light chain having CDR1, 2and/or 3 sequences according to the respective formula selected fromFormulas (I) to (VIII). In any embodiment herein, a particular HCDR1-3or LCDR-1-3 may be specified as having a sequence of Formulas (I) to(VIII). In one preferred embodiment, the antibody comprises a lightchain comprising the three LCDRs and a heavy chain comprising the threeHCDRs.

In one embodiment, HCDR1 comprises an amino acid sequence of Formula(I):

(SEQ ID NO: 261) G-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅,

wherein Xaa₁ may be. Phe or Tyr, Xaa₂ may be Thr or Ser, Xaa₃ may beIle, Leu or Phe, Xaa₄ may be Ser or Thr and Xaa₅ may be Asn, Tyr, Ser,Thr or Arg. Optionally any 1, 2 or 3 of said Xaa₁₋₅ may be aconservative or non-conservative substitution of any of the amino acidsindicated or a deletion or insertion.

In one embodiment, HCDR1 comprises an amino acid sequence of Formula(II):

(SEQ ID NO: 262) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅,

wherein Xaa₁ may be. Asn, Ser, Thr or Arg, Xaa₂ may be Gly, Asp, Ser orTyr, Xaa₃ may be Thr, Tyr, Ala, Gly, Trp, Xaa₄ may be Ser, Ala or Met,and Xaa₅ may be His, Trp, Gln, Ser or Asn. Optionally any 1, 2 or 3 ofsaid Xaa₁₋₅ may be a conservative or non-conservative substitution ofany of the amino acids indicated or a deletion or insertion.

In one embodiment, HCDR1 comprises an amino acid sequence of Formula(III):

(SEQ ID NO: 263) Xaa₁-Y-Xaa₂-M-Xaa₃,

wherein Xaa₁₋₃ may each be a conservative or non-conservativesubstitution of any of the amino acids indicated or a deletion orinsertion, wherein Xaa₁ may be Asn, Ser, Thr or Arg, Xaa₂ may be Thr,Tyr, Ala, Gly, Trp, and Xaa₃ may be His, Trp, Gln, Ser or Asn.

In one embodiment, HCDR2 comprises an amino acid sequence of Formula(IV):

(SEQ ID NO: 264) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀,

wherein Xaa₁ may be Phe, Thr, His, Asn, Tyr or Met, Xaa₂ may be Val orIle, Xaa₃ may be Ser, Phe, His, Asn or Trp, Xaa₄ may be Arg, Tyr, Ser,Trp or Pro, Xaa₅ may be Gly, Asp or Ser, Xaa₆ may be Thr, Gly, Asp orSer, Xaa, may be Asn, Thr, Ser, Asp, Arg or Gly, Xaa₆ may be Tyr, Lys,Glu, Arg, Ile or Thr, Xaa₉ may be Ile, Thr, Tyr, Asn or Asp, and Xaa₁₀may be Tyr, Arg or Glu. Optionally any 1, 2, 3 or 4 of said Xaa₁₋₁₀ maybe a conservative or non-conservative substitution of any of the aminoacids indicated or a deletion or insertion.

In one embodiment, HCDR3 comprises an amino acid sequence of Formula(V):

(SEQ ID NO: 265) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉,

wherein Xaa₁ may be Ile, Pro, Arg, Gly, Asp or Glu, Xaa₂ may be Ser,Tyr, Thr, Asn, Asp, Leu, Arg, Glu or Met, Xaa₃ may be Asp, Glu, Trp,Gln, Phe, Asn or Thr, Xaa₄ may be Tyr, Glu, Gly, Phe, Trp, His or Pro,Xaa₅ may be Asp, Arg, Tyr, Thr, Gly or Trp, Xaa₆ may be Gly, Tyr, Thr,Phe, Val, Met or Asn, Xaa, may be Ala, Phe, Asp, Met or Leu, Xaa may beTrp, Tyr, Asp or Glu, and Xaa₉ may be Leu, Tyr, Asp, Phe or Val.Optionally any 1, 2, 3 or 4 of said Xaa₁₋₉ may be a conservative ornon-conservative substitution of any of the amino acids indicated or adeletion or insertion.

In one embodiment, LCDR1 comprises an amino acid sequence of Formula(VI):

(SEQ ID NO: 266) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁,

wherein Xaa₁ may be Ser, Lys or Arg, Xaa₂ may be Ser or Ala, Xaa₃ may beThr or Ser, Xaa₄ may be Ser or Gln, Xaa₅ may be Asn or Ser, Xaa₆ may beVal, Leu or Ile, Xaa, may be Asp, Asn, Val, Leu, Gly or Ser, Xaa₆ may beTyr, Ser, Asn, Thr or Asp, Xaa₉ may be Asp, Met, Ile, Ser or Tyr, Xaa₁₀may be Val, His, Tyr, Ser, Ile, or Leu, and Xaa₁₁ may be Ala, Phe, Asnor His. Optionally any 1, 2, 3 or 4 of said Xaa₁₋₁₁ may be aconservative or non-conservative substitution of any of the amino acidsindicated, or a deletion or insertion.

In one embodiment, LCDR2 comprises an amino acid sequence of Formula(VII):

(SEQ ID NO: 267) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇,

wherein Xaa₁ may be Ser, Lys, Arg, Phe, Tyr or Ala, Xaa₂ may be Ile,Thr, Ala or Ser, Xaa₃ may be Ser or Glu, Xaa₄ may be Lys, Glu, Asn, Thr,Gln or Ser, Xaa₅ may be Leu, Arg, Ser or Ile, Xaa₆ may be Tyr, Phe, Ala,Glu, Ile or Ser, and Xaa, may be Thr, Ser or Gly. Optionally any 1, 2, 3or 4 of said Xaa₁₋₇ may be a conservative or non-conservativesubstitution of any of the amino acids indicated or a deletion orinsertion.

In one embodiment, LCDR3 comprises an amino acid sequence of Formula(VIII):

(SEQ ID NO: 268) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉,

wherein Xaa₁ may be Phe or Gln, Xaa₂ may be His, Asn or Gln, Xaa₃ may beSer, Gly, His, Trp or Arg, Xaa₄ may be Asn, His, Tyr, Thr or Ser, Xaa₅may be Phe, Ser, Thr, His, Ile or Tyr, Xaa₆ may be Trp, Phe, Thr, Ile,Val, Asn or Try, Xaa, is Pro, Xaa may be Phe, Trp, Pro or Leu, and Xaa₉is Thr. Optionally any 1, 2, 3 or 4 of said Xaa₁₋₉ may be a conservativeor non-conservative substitution of any of the amino acids indicated ora deletion or insertion.

In one embodiment, an antibody of the invention may comprise a lightchain comprising:

-   -   a a light chain CDR1 (LCDR1) comprising an amino acid sequence        of SEQ ID NO: 266; and/or    -   b a light chain CDR2 (LCDR2) comprising an amino acid sequence        of SEQ ID NO: 267; and/or    -   c a light chain CDR3 (LCDR3) comprising an amino acid sequence        of SEQ ID NO: 268.

In one embodiment, an antibody of the invention may comprise a heavychain comprising:

-   -   d a heavy chain CDR1 (HCDR1) comprising an amino acid sequence        selected from SEQ ID NOS: 261, 262 and 263; and/or    -   e a heavy chain CDR2 (HCDR2) comprising an amino acid sequence        of SEQ ID NO: 264; and/or    -   f a heavy chain CDR3 (HCDR3) comprising an amino acid sequence        of SEQ ID NO: 265.

Fragments and Derivatives

Fragments and derivatives of antibodies of this invention (which areencompassed by the term “antibody” or “antibodies” as used in thisapplication, unless otherwise stated or clearly contradicted bycontext), preferably a 6E4, 20C6, 16A8, 9C10, 19E9, 12A10, 10A7, 18E8,10F3, 15F9 or 14B4-like antibody, can be produced by techniques that areknown in the art. “Fragments” comprise a portion of the intact antibody,generally the antigen binding site or variable region. Examples ofantibody fragments include Fab, Fab′, Fab′-SH, F (ab′) 2, and Fvfragments; diabodies; any antibody fragment that is a polypeptide havinga primary structure consisting of one uninterrupted sequence ofcontiguous amino acid residues (referred to herein as a “single-chainantibody fragment” or “single chain polypeptide”), including withoutlimitation (1) single-chain Fv molecules (2) single chain polypeptidescontaining only one light chain variable domain, or a fragment thereofthat contains the three CDRs of the light chain variable domain, withoutan associated heavy chain moiety and (3) single chain polypeptidescontaining only one heavy chain variable region, or a fragment thereofcontaining the three CDRs of the heavy chain variable region, without anassociated light chain moiety; and multispecific antibodies formed fromantibody fragments. Included, inter alia, are a nanobody, domainantibody, single domain antibody or a “dAb”.

Fragments of the present antibodies can be obtained using standardmethods. For instance, Fab or F (ab′) 2 fragments may be produced byprotease digestion of the isolated antibodies, according to conventionaltechniques. It will be appreciated that immunoreactive fragments can bemodified using known methods, for example to slow clearance in vivo andobtain a more desirable pharmacokinetic profile the fragment may bemodified with polyethylene glycol (PEG).

Alternatively, the DNA of a hybridoma producing an antibody of theinvention may be modified so as to encode a fragment of the invention.The modified DNA is then inserted into an expression vector and used totransform or transfect an appropriate cell, which then expresses thedesired fragment.

In certain embodiments, the DNA of a hybridoma producing an antibody ofthis invention can be modified prior to insertion into an expressionvector, for example, by substituting the coding sequence for humanheavy- and light-chain constant domains in place of the homologousnon-human sequences, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of theoriginal antibody. Typically, such non-immunoglobulin polypeptides aresubstituted for the constant domains of an antibody of the invention.

Thus, according to another embodiment, the antibody of this invention ishumanized. “Humanized” forms of antibodies according to this inventionare specific chimeric immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′, F (ab′) 2, or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from the murine immunoglobulin. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of the original antibody(donor antibody) while maintaining the desired specificity, affinity,and capacity of the original antibody.

In some instances, Fv framework residues of the human immunoglobulin maybe replaced by corresponding non-human residues. Furthermore, humanizedantibodies can comprise residues that are not found in either therecipient antibody or in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof the original antibody and all or substantially all of the FR regionsare those of a human immunoglobulin consensus sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature, 321, pp.522 (1986); Reichmann et al, Nature, 332, pp. 323 (1988); Presta, Curr.Op. Struct. Biol., 2, pp. 593 (1992); Verhoeyen et Science, 239, pp.1534; and U.S. Pat. No. 4,816,567, the entire disclosures of which areherein incorporated by reference.)

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of an antibody of this invention is screenedagainst the entire library of known human variable-domain sequences. Thehuman sequence which is closest to that of the mouse is then accepted asthe human framework (FR) for the humanized antibody (Sims et al., J.Immunol. 151, pp. 2296 (1993); Chothia and Lesk, J. Mol. 196, 1987, pp.901). Another method uses a particular framework from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework can be used for several differenthumanized antibodies (Carter et al., PNAS 89, pp. 4285 (1992); Presta etal., J. Immunol., 151, p. 2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for MICA receptors and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional structures ofselected candidate immunoglobulin sequences. Inspection of thesedisplays permits analysis of the likely role of the residues in thefunctioning of the candidate immunoglobulin sequence, i.e., the analysisof residues that influence the ability of the candidate immunoglobulinto bind its antigen. In this way, FR residues can be selected andcombined from the consensus and import sequences so that the desiredantibody characteristic, such as increased affinity for the targetantigen (s), is achieved. In general, the CDR residues are directly andmost substantially involved in influencing antigen binding.

Another method of making “humanized” monoclonal antibodies is to use aXenoMouse (Abgenix, Fremont, Calif.) as the mouse used for immunization.A XenoMouse is a murine host according to this invention that has hadits immunoglobulin genes replaced by functional human immunoglobulingenes. Thus, antibodies produced by this mouse or in hybridomas madefrom the B cells of this mouse, are already humanized. The XenoMouse isdescribed in U.S. Pat. No. 6,162,963, which is herein incorporated inits entirety by reference.

Human antibodies may also be produced according to various othertechniques, such as by using, for immunization, other transgenic animalsthat have been engineered to express a human antibody repertoire(Jakobovitz et al., Nature 362 (1993) 255), or by selection of antibodyrepertoires using phage display methods. Such techniques are known tothe skilled person and can be implemented starting from monoclonalantibodies as disclosed in the present application.

The antibodies of the present invention may also be derivatized to“chimeric” antibodies (immunoglobulins) in which a portion of theheavy/light chain(s) is identical with or homologous to correspondingsequences in the original antibody, while the remainder of the chain (s)is identical with or homologous to corresponding sequences in antibodiesderived from another species or belonging to another antibody class orsubclass, as well as fragments of such antibodies, so long as theyexhibit the desired biological activity and binding specificity (Cabillyet. al., supra; Morrison et al., Proc. Natl. Acad. Sci. U.S.A., pp. 6851(1984)).

The invention provides anti-MICA antibody molecules which are directedto and, in embodiments, are internalized into cells. They are capable ofdelivering therapeutic agents or detectable agents to or into cellsexpressing MICA, but not to or into cells where MICA polypeptides arenot expressed. Thus, the invention also provides anti-MICAimmunoconjugates comprising an anti-MICA antibody as described herein,which is conjugated to a therapeutic agent or a detectable agent (or anyother moiety that serves as a payload of interest to be delivered to aMICA-expressing cell. In embodiments, the affinity for MICA of ananti-MICA immunoconjugate is at least 10, 25, 50, 75, 80, 90, or 95% ofthat for the unconjugated antibody. This can be determined using cellsurface MICA or isolated MICA.

Useful detectable agents with which an antibody or an antibody portionof the invention may be derivatized (or labeled) include fluorescentcompounds, various enzymes, prosthetic groups, luminescent materials,bioluminescent materials, fluorescent emitting metal atoms, e.g.,europium (Eu), and other anthanides, and radioactive materials(described above). Exemplary fluorescent detectable agents includefluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-I-napthalenesulfonyl chloride, phycoerythrin and thelike. An antibody may also be derivatized with detectable enzymes, suchas alkaline phosphatase, horseradish peroxidase, β-galactosidase,acetylcholinesterase, glucose oxidase and the like. When an antibody isderivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with a prosthetic group(e.g., streptavidin/biotin and avidin/biotin). For example, an antibodymay be derivatized with biotin, and detected through indirectmeasurement of avidin or streptavidin binding. Examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of bioluminescent materials include luciferase,luciferin, and aequorin. Alternatively, the anti-MICA antibody may beassociated with a second antibody that binds to the anti-MICA antibody,wherein the second antibody is derivatized with a detectable label;binding said second antibody into contact with the anti-MICA antibody,in vitro or in vivo, will allow the anti-MICA to serve as a labeledantibody.

Conjugation to a detectable moiety is useful, inter alia, when anantibody of the invention is used for diagnostic purposes. Such purposesinclude, but are not limited to, assaying biological samples; e.g., ablood sample or tissue biopsy, for the presence of MICA-expressingcells, and detecting the presence, level, or activity of MICA-expressingcells in an individual. Such assay and detection methods can be used inthe diagnostic/therapeutic methods of the invention, e.g., involvingdetecting MICA expression in cells of a patient and if the patient'scells are determined to express MICA, subsequently administering a MICAmodulating antibody of the invention.

In certain embodiments, the present antibodies are used to purifyMICA-expressing cells from a biological sample. Biological samples canbe obtained from a patient, e.g. for diagnostic or ex vivo therapeuticpurposes, or from individuals or non-human primates to obtain a sourceof such cells for research purposes.

In one such embodiment, labeled antibodies of the invention can be usedin FACS sorting to purify or isolate MICA-expressing cells from abiological sample. Alternatively, in some embodiments conjugation of anantibody of this invention to a solid support can be useful as a toolfor affinity purification of cells bearing a MICA receptor on their cellsurface from a biological sample, such as a blood sample or cells from atissue biopsy from an individual. This method of purification is anotheralternate embodiment of the present invention, as is the resultingpurified population of cells.

Regardless of the method used to isolate or purify the MICA-expressingcells, the ability to do so is useful for numerous purposes, e.g. todiagnose a MICA-associated disorder by assessing the number or activityof MICA-expressing cells, e.g., prior to administration of anti-MICAantibodies as described herein. Further, purified MICA-expressing cellsare useful in a research context, e.g., to better characterize the cellsand their various properties and behaviors, as well as to identifycompounds or methods that can be used to modulate their behavior,activity, survival, or proliferation.

Modified Constant Regions

In view of the ability of the anti-MICA antibodies of the invention toinduce ADCC and CDC, the antibodies of the invention can also be madewith modifications that increase their ability to bind Fc receptorswhich can affect effector functions such as antibody-dependentcytotoxicity, mast cell degranulation, and phagocytosis, as well asimmunomodulatory signals such as regulation of lymphocyte proliferationand antibody secretion. Typical modifications include modified humanIgG1 constant regions comprising at least one amino acid modification(e.g. substitution, deletions, insertions), and/or altered types ofglycosylation, e.g., hypofucosylation. Such modifications can affectinteraction with Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII(CD 16). FcγRI (CD64), FcγRIIA (CD32A) and FcγRIII (CD 16) areactivating (i.e., immune system enhancing) receptors while FcγRIIB(CD32B) is an inhibiting (i.e., immune system dampening) receptor. Amodification may, for example, increase binding of the Fc domain toFcγRIIIa on effector (e.g. NK) cells.

Anti-MICA antibodies preferably comprise an Fc domain (or portionthereof) of human IgG1 or IgG3 isotype, optionally modified. The aminoacid sequence of positions 230 to 447 sequence of a human IgG1 Fc region(GenBank accession #: J00228) is shown as follows:

(SEQ ID NO: 134) PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK;

Residues 230-341 (Kabat EU) are the Fc CH2 region. Residues 342-447(Kabat EU) are the Fc CH3 region. Anti-MICA antibodies may comprise avariant Fc region having one or more amino acid modifications (e.g.,substitutions, deletions, insertions) in one or more portions, whichmodifications increase the affinity and avidity of the variant Fc regionfor an FcγR (including activating and inhibitory FcγRs). In someembodiments, said one or more amino acid modifications increase theaffinity of the variant Fc region for FcγRIIIA and/or FcγRIIA. Inanother embodiment, the variant Fc region further specifically bindsFcγRIIB with a lower affinity than does the Fc region of the comparableparent antibody (i.e., an antibody having the same amino acid sequenceas the antibody of the invention except for the one or more amino acidmodifications in the Fc region). For example, the one or both of thehistidine residues at amino acid positions 310 and 435 may besubstituted, for example by lysine, alanine, glycine, valine, leucine,isoleucine, proline, methionine, tryptophan, phenylalanine, serine orthreonine (see, e.g. PCT publication no. WO 2007/080277); suchsubstituted constant regions provide decreased binding to the inhibitoryFcγRIIB without decreasing binding to the activatory FcγRIIIA. In someembodiments, such modifications increase the affinity of the variant Fcregion for FcγRIIIA and/or FcγRIIA and also enhance the affinity of thevariant Fc region for FcγγRIIB relative to the parent antibody. In otherembodiments, said one or more amino acid modifications increase theaffinity of the variant Fc region for FcγRIIIA and/or FcγRIIA but do notalter the affinity of the variant Fc regions for FcγRIIB relative to theFc region of the parent antibody. In another embodiment, said one ormore amino acid modifications enhance the affinity of the variant Fcregion for FcγRIIIA and FcγRIIA but reduce the affinity for FcγRIIBrelative to the parent antibody. Increased affinity and/or avidityresults in detectable binding to the FcγR or FcγR-related activity incells that express low levels of the FcγR when binding activity of theparent molecule (without the modified Fc region) cannot be detected inthe cells.

In one embodiment, said one or more modifications to the amino acids ofthe Fc region reduce the affinity and avidity of the antibody for one ormore FcγR receptors. In a specific embodiment, the invention encompassesantibodies comprising a variant Fc region, wherein said variant Fcregion comprises at least one amino acid modification relative to a wildtype Fc region, which variant Fc region only binds one FcγR, whereinsaid FcγR is FcγRIIIA or FcγRIIA.

The affinities and binding properties of the molecules, e.g.,antibodies, of the invention for an FcγR can be determined using invitro assays (biochemical or immunological based assays) known in theart for determining antibody-antigen or Fc-FcγR interactions, i.e.,specific binding of an antigen to an antibody or specific binding of anFc region to an FcγR, respectively, including but not limited to ELISAassay, surface plasmon resonance assay, immunoprecipitation assays.

In some embodiments, the molecules of the invention comprising a variantFc region comprise at least one amino acid modification (for example,possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications)in the CH3 domain of the Fc region. In other embodiments, the moleculesof the invention comprising a variant Fc region comprise at least oneamino acid modification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8,9, or more amino acid modifications) in the CH2 domain of the Fc region,which is defined as extending from amino acids 231-341. In someembodiments, the molecules of the invention comprise at least two aminoacid modifications (for example, possessing 2, 3, 4, 5, 6, 7, 8, 9, ormore amino acid modifications), wherein at least one such modificationis in the CH3 region and at least one such modification is in the CH2region. The invention further encompasses amino, acid modification inthe hinge region. In a particular embodiment, the invention encompassesamino acid modification in the CH1 domain of the Fc region, which isdefined as extending from amino acids 216-230.

Any combination of Fc modifications can be made, for example anycombination of different modifications disclosed in U.S. Pat. Nos.7,632,497; 7,521,542; 7,425,619; 7,416,727; 7,371,826; 7,355,008;7,335,742; 7,332,581; 7,183,387; 7,122,637; 6,821,505 and 6,737,056; inPCT Publications Nos. WO2011/109400; WO 2008/105886; WO 2008/002933; WO2007/021841; WO 2007/106707; WO 06/088494; WO 05/115452; WO 05/110474;WO 04/1032269; WO 00/42072; WO 06/088494; WO 07/024249; WO 05/047327; WO04/099249 and WO 04/063351; and in Presta, L. G. et al. (2002) Biochem.Soc. Trans. 30(4):487-490; Shields, R. L. et al. (2002) J. Biol. Chem.26; 277(30):26733-26740 and Shields, R. L. et al. (2001) J. Biol. Chem.276(9):6591-6604).

The invention encompasses anti-MICA antibodies a which comprise avariant Fc region, wherein the variant Fc region comprises at least oneamino acid modification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8,9, or more amino acid modifications) relative to a wild-type Fc region,such that the molecule has an enhanced effector function relative to amolecule comprising a wild-type Fc region, optionally wherein thevariant Fc region comprises a substitution at any one or more ofpositions 221, 243, 247, 255, 256, 258, 267, 268, 269, 270, 272, 276,278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300,301, 303, 305, 307, 308, 309, 310, 311, 312, 316, 320, 322, 326, 329,330, 332, 331, 333, 334, 335, 337, 338, 339, 340, 359, 360, 370, 373,376, 378, 392, 396, 399, 402, 404, 416, 419, 421, 430, 434, 435, 437,438 and/or 439.

The invention encompasses anti-MICA antibodies a which comprise avariant Fc region, wherein the variant Fc region comprises at least oneamino acid modification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8,9, or more amino acid modifications) relative to a wild-type Fc region,such that the molecule has an enhanced effector function relative to amolecule comprising a wild-type Fc region, optionally wherein thevariant Fc region comprises a substitution at any one or more ofpositions 329, 298, 330, 332, 333 and/or 334 (e.g. S239D, S298A, A330L,1332E, E333A and/or K334A substitutions).

In one embodiment, antibodies having variant or wild-type Fc regions mayhave altered glycosylation patterns that increase Fc receptor bindingability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP1,176,195; PCT Publications WO 06/133148; WO 03/035835; WO 99/54342,each of which is incorporated herein by reference in its entirety.

Generally, such antibodies with altered glycosylation are“glyco-optimized” such that the antibody has a particular N-glycanstructure that produces certain desireable properties, including but notlimited to, enhanced ADCC and effector cell receptor binding activitywhen compared to non-modified antibodies or antibodies having anaturally occurring constant region and produced by murine myeloma NSOand Chinese Hamster Ovary (CHO) cells (Chu and Robinson, Current OpinionBiotechnol. 2001, 12: 180-7), HEK293T-expressed antibodies as producedherein in the Examples section, or other mammalian host cell linescommonly used to produce recombinant therapeutic antibodies.

Monoclonal antibodies produced in mammalian host cells contain anN-linked glycosylation site at Asn297 of each heavy chain. Glycans onantibodies are typically complex biatennary structures with very low orno bisecting N-acetylglucosamine (bisecting GlcNAc) and high levels ofcore fucosylation. Glycan temini contain very low or no terminal sialicacid and variable amounts of galactose. For a review of effects ofglycosylation on antibody function, see, e.g., Wright & Morrison, TrendBiotechnol. 15:26-31 (1997). Considerable work shows that changes to thesugar composition of the antibody glycan structure can alter Fc effectorfunctions. The important carbohydrate structures contributing toantibody activity are believed to be the fucose residues attached viaalpha-1,6 linkage to the innermost N-acetylglucosamine (GlacNAc)residues of the Fc region N-linked oligosaccharides (Shields et al.,2002).

FcγR binding requires the presence of oligosaccharides covalentlyattached at the conserved Asn297 in the Fc region of human IgGI, IgG2 orIgG3 type. Non-fucosylated oligosaccharides structures have recentlybeen associated with dramatically increased in vitro ADCC activity. “Asn297” according to the invention means amino acid asparagine located atabout position 297 in the Fc region; based on minor sequence variationsof antibodies, Asn297 can also be located some amino acids (usually notmore than +3 amino acids) upstream or downstream.

Historically, antibodies produced in CHO cells contain about 2 to 6% inthe population that are nonfucosylated. YB2/0 (rat myeloma) and Lecl3cell line (a lectin mutant of CHO line which has a deficient GDP-mannose4,6-dehydratase leading to the deficiency of GDP-fucose or GDP sugarintermediates that are the substrate of alpha6-fucosyltransferase havebeen reported to produce antibodies with 78 to 98% non-fucosylatedspecies. In other examples, RNA interference (RNAi) or knock-outtechniques can be employed to engineer cells to either decrease the FUT8mRNA transcript levels or knock out gene expression entirely, and suchantibodies have been reported to contain up to 70% non-fucosylatedglycan.

The invention comprises an antibody binding to MICA being glycosylatedwith a sugar chain at Asn297, said antibody showing increased bindingaffinity via its Fc portion to FcγRIII. In one embodiment of theinvention, an antibody will comprise a constant region comprising atleast one amino acid alteration in the Fc region that improves antibodybinding to FcγRIIIa and/or ADCC.

In one aspect, the antibodies of the invention are hypofucosylated intheir constant region. Such antibodies may comprise an amino acidalteration or may not comprise an amino acid alteration but be producedor treated under conditions so as to yield such hypofucosylation. In oneaspect, an antibody composition of the invention comprises a chimeric,human or humanized antibody described herein, wherein at least 20, 30,40, 50, 60, 75, 85, 90, 95% or substantially all of the antibody speciesin the composition have a constant region comprising a core carbohydratestructure (e.g. complex, hybrid and high mannose structures) which lacksfucose. In one embodiment, provided is an antibody composition which isfree of antibodies comprising a core carbohydrate structure havingfucose. The core carbohydrate will preferably be a sugar chain atAsn297.

In one embodiment, the invention comprises an antibody composition ofthe invention, e.g. a composition comprising antibodies which bind toMICA, are glycosylated with a sugar chain at Asn297, wherein theantibodies are partially fucosylated. Partially fucosylated antibodiesare characterized in that the proportion of anti-MICA antibodies in thecomposition that lack fucose within the sugar chain at Asn297 is between20% and 90%, preferably between 20% and 80%, preferably between 20% and50%, 55%, 60%, 70% or 75%, between 35% and 50%, 55%, 60%, 70% or 75%, orbetween 45% and 50%, 55%, 60%, 70% or 75%. Preferably the antibody is ofhuman IgGI or IgG3 type.

The sugar chain show can further show any characteristics (e.g. presenceand proportion of complex, hybrid and high mannose structures),including the characteristics of N-linked glycans attached to Asn297 ofan antibody from a human cell, or of an antibody recombinantly expressedin a rodent cell, murine cell (e.g. CHO cell) or in an avian cell. Inone embodiment, the antibody is expressed in a cell that is lacking in afucosyltransferase enzyme such that the cell line produces proteinslacking fucose in their core carbohydrates. For example, the cell linesMs704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha(1,6) fucosyltransferase), such that antibodies expressed in the Ms704,Ms705, and Ms709 cell lines lack fucose on their core carbohydrates.These cell lines were created by the targeted disruption of the FUT8gene in CHO/DG44 cells using two replacement vectors (see U.S. PatentPublication No. 20040110704 by Yamane et al.; and Yamane-Ohnuki et al.(2004) Biotechnol Bioeng 87:614-22, the disclosures of which areincorporated herein by reference). Other examples have included use ofantisense suppression, double-stranded RNA (dsRNA) interference, hairpinRNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA)interference to functionally disrupt the FUT8 gene. In one embodiment,the antibody is expressed in a cell line with a functionally disruptedFUT8 gene, which encodes a fucosyl transferase, such that antibodiesexpressed in such a cell line exhibit hypofucosylation by reducing oreliminating the alpha 1,6 bond-related enzyme.

In one embodiment, the antibody is expressed in cell lines engineered toexpress glycoprotem-modifying glycosyl transferases (e.g.,beta(1,4)-N-acetylglucosaminyltransferase III (GnTHI)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (PCT Publication WO 99/54342 by Umana et al.; and Umanaet al. (1999) Nat. Biotech. 17:176-180, the disclosures of which areincorporated herein by reference).

In another embodiment, the antibody is expressed and the fucosylresidue(s) is cleaved using a fucosidase enzyme. For example, thefucosidase alpha-L-fucosidase removes fucosyl residues from antibodies(Tarentino, et al. (1975) Biochem. 14:5516-5523). In other examples, acell line producing an antibody can be treated with a glycosylationinhibitor; Zhou et al. Biotech. and Bioengin. 99: 652-665 (2008)described treatment of CHO cells with the alpha-mannosidase I inhibitor,kifunensine, resulting in the production of antibodies withnon-fucosylated oligomannose-type N-glucans.

In one embodiment, the antibody is expressed in a cell line whichnaturally has a low enzyme activity for adding fucosyl to theN-acetylglucosamine that binds to the Fc region of the antibody or doesnot have the enzyme activity, for example the rat myeloma cell lineYB2/0 (ATCC CRL 1662). Other example of cell lines include a variant CHOcell line, Led 3 cells, with reduced ability to attach fucosyl toAsn(297)-linked carbohydrates, also resulting in hypofucosylation ofantibodies expressed in that host cell (WO 03/035835 (Presta et al); andShields, R X. et al. (2002) J. Biol. Chem. 277:26733-26740, thedisclosures of which are incorporated herein by reference). In anotherembodiment, the antibody is expressed in an avian cell, preferably aEBx® cell (Vivalis, France) which naturally yields antibodies with lowfucose content e.g WO2008/142124. Hypofucosylated glycans can also beproduced in cell lines of plant origin, e.g. WO 07/084926A2 (BiolexInc.), WO 08/006554 (Greenovation Biotech GMBH), the disclosures ofwhich are incorporated herein by reference.

Uses in Diagnostics and Therapy

In certain embodiments, the present antibodies are used to purify oridentify MICA positive cells from a biological sample. Biologicalsamples can be obtained from a patient, e.g. for diagnostic or ex vivotherapeutic purposes, or from individuals or non-human primates toobtain a source of such cells for research purposes.

MICA positive cells can be purified or identified using the presentantibodies with any of a number of standard methods. For example,peripheral blood cells can be sorted using a FACS scanner using labeledantibodies specific for MICA, and optionally to other cell surfacemolecules typically present on cells.

In addition, the antibodies of the invention can be conjugated orcovalently linked to a solid support and used to purify or identify MICApositive cells or any cells expressing MICA from a biological sample,e.g., from a blood sample or tissue biopsy from a patient or otherindividual. Specifically, the biological sample is placed into contactwith the antibodies under conditions that allow cells within the sampleto bind to the antibody, and then the cells are eluted from thesolid-support-bound antibody.

Regardless of the method used to isolate, purify or identify the MICApositive cells, the ability to do so is useful for numerous purposes;e.g. to diagnose a disorder characterized by a pathogenic expansion ofMICA-expressing cells, by assessing the number or activity or othercharacteristics of MICA positive cells obtained from a patient, or toevaluate the ability of the antibodies of the invention, or fragments orderivatives thereof, to modulate the activity or behavior of the cellsof a patient prior, e.g., to one of the herein-described treatmentsusing the antibodies. Further, purified MICA positive cells are usefulin a research context, e.g., to better characterize the cells and theirvarious properties and behaviors, as well as to identify compounds ormethods that can be used to modulate their behavior, activity, orproliferation. The antibodies of the invention can also be useful indiagnostic methods, for example in methods of detecting MICApolypeptides on cells, e.g. disease cells from a patient.

The present invention also provides pharmaceutical compositions thatcomprise an antigen-binding agent (e.g. an antibody) according to theinvention which specifically binds to MICA polypeptides on the surfaceof cells. The antibody preferably inhibits the growth or activity of thecells and/or leads to the elimination of the MICA positive cells,preferably via induction of CDC and/or ADCC. The composition furthercomprises a pharmaceutically acceptable carrier.

The invention further provides a method of inhibiting the growth oractivity of, and/or depleting, MICA-positive cells, in a patient in needthereof, comprising the step of administering to said patient acomposition according to the invention. Such treatment methods can beused for a number of disorders, including, but not limited to thetreatment of cancers.

As demonstrated herein, non-blocking anti-MICA antibodies areparticularly effective at inducing lysis of MICA-expressing cells byeffector cells. The antibodies have a dual mode of action, by bindingMICA and engaging activating Fcγ receptors on effector cells they inducelysis by effector cells, and by not blocking NKG2D signaling theyprevent a neutralization of NKG2D-mediated activation of NK cellreactivity, or by blocking NKG2D signaling and reducing sMICA-inducedNKG2D downmodulation. The antibodies preferably comprise human heavychain constant regions sequences that lead to the depletion ofMICA-expression cells (e.g. tumor cells) to which they are bound andpreferably comprise an Fc portion that induces CDC and/or ADCC. Thecomposition further comprises a pharmaceutically acceptable carrier.Such compositions are also referred to as “antibody compositions” of theinvention. In one embodiment, antibody compositions of this inventioncomprise an antibody disclosed in the antibody embodiments above.

In one aspect, the methods of treatment of the invention compriseadministering to an individual a composition comprising anantigen-binding compound that binds MICA in a therapeutically effectiveamount. A therapeutically effective amount may be for example ansufficient to cause an increase in the depletion of MICA cells in vivo,or an increase in the frequency of activated, reactive, cytotoxic and/orIFN_(γ)-production of NKG2D+ effector cells (e.g. NK cells) towardsMICA-expressing tumor cells.

The methods of the present invention are utilized advantageously for thetreatment of cancers and other proliferative diseases including, but notlimited to, carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroidand skin, including squamous cell carcinoma; hematopoietic tumors oflymphoid lineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkinslymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including neuroblastoma and glioma; tumors of the central andperipheral nervous system, including astrocytoma, neuroblastoma, glioma,and schwannomas; tumors of mesenchymal origin, including fibrosarcoma,rhabdomyoscaroma, and osteosarcoma; and other tumors, includingmelanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroidfollicular cancer and teratocarcinoma. Other exemplary disorders thatcan be treated according to the invention include hematopoietic tumorsof lymphoid lineage, for example T-cell and B-cell tumors, including butnot limited to T-cell disorders such as T-prolymphocytic leukemia(T-PLL), including of the small cell and cerebriform cell type; largegranular lymphocyte leukemia (LGL) preferably of the T-cell type; Sezarysyndrome (SS); Adult T-cell leukemia lymphoma (ATLL); a/d T-NHLhepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma(pleomorphic and immunoblastic subtypes); angio immunoblastic T-celllymphoma; angiocentric (nasal) T-cell lymphoma; anaplastic (Ki 1+) largecell lymphoma; intestinal T-cell lymphoma; T-lymphoblastic; andlymphoma/leukaemia (T-Lbly/T-ALL).

In some embodiments, prior to the administration of the anti-MICAantibody or composition, the presence of MICA on cells (e.g. tumorcells) of the patient will be assessed, e.g., to determine the relativelevel and activity of MICA-positive cells in the patient as well as toconfirm the binding efficacy of the antibodies to the cells of thepatient. A patient whose tumor cells express MICA can then be treatedwith an anti-MICA antibody or composition. This can be accomplished byobtaining a sample of PBLs or tumor cells from the site of the disorder,and testing e.g., using immunoassays, to determine the relativeprominence of MICA and optionally further other markers on the cells.Other methods can also be used to detect expression of MICA and othergenes, such as RNA-based methods, e.g., RT-PCR or Northern blotting.

In one embodiment, where it is sought to inhibit the activity or growthof, or deplete, a patient's MICA-positive cells, the ability of theanti-MICA antibody to inhibit proliferation of or deplete a patient'sMICA-positive cells is assessed. If the MICA-positive cells are depletedby the anti-MICA antibody or composition, the patient is determined tobe responsive to therapy with an anti-MICA antibody or composition, andoptionally the patient is treated with an anti-MICA antibody orcomposition.

The treatment may involve multiple rounds of antibody or compoundadministration. For example, following an initial round ofadministration, the level and/or activity of MICA-expressing cells(e.g., on malignant tumor cells), in the patient will generally bere-measured, and, if still elevated, an additional round ofadministration can be performed. In this way, multiple rounds of MICAdetection and antibody or compound administration can be performed,e.g., until the disorder is brought under control.

In some embodiments, the method may comprise the additional step ofadministering to said patient an appropriate additional (second)therapeutic agent selected from an immunomodulatory agent, a hormonalagent, a chemotherapeutic agent, or a second antibody (e.g. a depletingantibody) that binds to a polypeptide present on a MICA-expressing cell.Such additional agents can be administered to said patient as a singledosage form together with said antibody, or as a separate dosage form.The dosage of the antibody (or antibody and the dosage of the additionaltherapeutic agent collectively) are sufficient to detectably induce,promote, and/or enhance a therapeutic response in the patient. Whereadministered separately, the antibody, fragment, or derivative and theadditional therapeutic agent are desirably administered under conditions(e.g., with respect to timing, number of doses, etc.) that result in adetectable combined therapeutic benefit to the patient.

For tumor (e.g. solid tumor) treatment, for example, the administrationof a composition of the present invention may be used in combinationwith classical approaches, such as surgery, radiotherapy, chemotherapy,and the like. The invention therefore provides combined therapies inwhich the present antibodies are used simultaneously with, before, orafter surgery or radiation treatment; or are administered to patientswith, before, or after conventional chemotherapeutic, radiotherapeuticor anti-angiogenic agents, or targeted immunotoxins or coaguligands.

Exemplary anti-cancer anti-angiogenic agents inhibit signaling by areceptor tyrosine kinase including but not limited to FGFR (fibroblastgrowth factor receptor, FGF-1,2), PDGFR (platelet derived growth factorreceptor), angiopoietins receptors (Ang-1,2), HGFR (hepatocytary growthfactor receptor), ephrines receptor (Eph), VEGFR1, VEGFR-2,3 PDGFR-α,PDGFR-β, CSF-1R, MET, Flt-3, c-Kit, bcr/abl, p38 alpha and FGFR-1.Further anti-angiogenic agents may include agents that inhibit one ormore of the various regulators of VEGF expression and production, suchas EGFR, flt-1, KDR, HER-2, COX-2, or HIF-1α. Another preferred class ofagents includes IMiD (immunomodulatory drugs), analogs derived fromthalidomide that have a wide range of effects, including both immune andnon-immune related effects. Representatives of the IMiD class includeCC-5013 (lenalidomide, Revlimid™), CC-4047 (Actimid™), and ENMD-0995.Another class of anti-angiogenic agent includes cilengitide (EMD 121974,integrin inhibitor), metalloproteinases (MPP) such as marinastat(BB-251). Another class of anti-angiogenic agents includes farnesylationinhibitors such as lonafarnib (Sarasar™), tipifarnib (Zarnestra™). Otheranti-angiogenic agents can also be suitable such as Bevacuzimab (mAb,inhibiting VEGF-A, Genentech); IMC-1121B (mAb, inhibiting VEGFR-2,ImClone Systems); CDP-791 (Pegylated DiFab, VEGFR-2, Celltech); 2C3(mAb, VEGF-A, Peregrine Pharmaceuticals); VEGF-trap (Soluble hybridreceptor VEGF-A, PIGF (placenta growth factor) Aventis/Regeneron).Another preferred class of agents includes the tyrosine kinase inhibitor(TKI) class, including, e.g., PTK-787 (TKI, VEGFR-1,-2, Vatalanib,Novartis); AEE788 (TKI, VEGFR-2 and EGFR, Novartis); ZD6474 (TKI,VEGFR-1,-2,-3, EGFR, Zactima, AstraZeneca); AZD2171 (TKI, VEGFR-1,-2,AstraZeneca); SU11248 (TKI, VEGFR-1,-2, PDGFR, Sunitinib, Pfizer);AG13925 (TKI, VEGFR-1,-2, Pfizer); AG013736 (TKI, VEGFR-1,-2, Pfizer);CEP-7055 (TKI, VEGFR-1,-2,-3, Cephalon); CP-547,632 (TKI, VEGFR-1,-2,Pfizer); GW786024 (TKI, VEGFR-1,-2,-3, GlaxoSmithKline); GW786034 (TKI,VEGFR-1,-2,-3, GlaxoSmithKline); sorafenib (TKI, Bay 43-9006,VEGFR-1,-2, PDGFR Bayer/Onyx); SU4312 (TKI, VEGFR, PDGFR, Pfizer),AMG706 (TKI, VEGFR-1,-2,-3, Amgen), XL647 (TKI, EGFR, HER2, VEGFR,ErbB4, Exelixis), XL999 (TKI, FGFR, VEGFR, PDGFR, Flt-3, Exelixis),PKC412 (TKI, KIT, PDGFR, PKC, FLT3, VEGFR-2, Novartis), AEE788 (TKI,EGFR, HER2, VEGFR, Novartis), OSI-930 (TKI, c-kit, VEGFR, OSIPharmaceuticals), OSI-817 (TKI, c-kit, VEGFR, OSI Pharmaceuticals), DMPQ(TKI, ERGF, PDGFR, erbB2, p56, pkA, pkC), MLN518 (TKI, FLT3, PDGFR,c-KIT, CT53518, Millennium Pharmaceuticals), lestaurinib (TKI, FLT3,CEP-701, Cephalon), ZD1839 (TKI, EGFR, gefitinib, Iressa, AstraZeneca),OSI-774 (TKI, EGFR, Erlotininb, Tarceva, OSI Pharmaceuticals), lapatinib(TKI, ErbB-2, EGFR, GD-2016, Tykerb, GlaxoSmithKline). Most preferredare tyrosine kinase inhibitors that inhibit one or more receptortyrosine kinases selected from the group consisting of VEGFR-1, VEGFR-2,VEGFR-3, PDGFR-α, β, Flt-3, c-Kit, p38 alpha, MET, c-RAF, b-RAF, bcr/abland FGFR-1.

In one embodiment, the second agent is a natural ligand of an effectorcell (e.g. NK cell) activating receptor or an antibody that binds andactivates an NK cell activating receptor other than NKG2D. In oneembodiment the agent is an agent that increases the presence of anatural ligand of an NK cell activating receptor other than NKG2D on thesurface of a target cell (e.g., infected cells, tumor cells,pro-inflammatory cells). NK cell activating receptors include, forexample, natural cytotoxicity receptors such as NKp30, NKp46, NKp44 oractivating KIR receptors (KIR2DS receptors, KIR2DS2, KIR2DS4). As usedherein, the term “activating NK receptor” refers to any molecule on thesurface of NK cells that, when stimulated, causes a measurable increasein any property or activity known in the art as associated with NKactivity, such as cytokine (for example IFN-γ and TNF-q production,increases in intracellular free calcium levels, the ability to targetcells in a redirected killing assay as described, e.g. elsewhere in thepresent specification, or the ability to stimulate NK cellproliferation. The term “activating NK receptor” includes but is notlimited to activating forms or KIR proteins (for example KIR2DSproteins), NKp30, NKp46, NKp44, NKG2D, IL-2R, IL-12R, IL-15R, IL-18R andIL-21R.

In one embodiment, the anti-cancer agent is a chemotherapeutic agent orradiation that upregulates expression of NKG2D ligands on the surface oftumor cells. This includes well known chemotherapies including ionizingand UV radiation, inhibitors of DNA replication, inhibitors of DNApolymerase, chromatin modifying treatments, as well as apoptosisinducing agents such as HDAC inhibitors trichostatin A and valproicacid. Preferred therapies are those that activate the DNA damageresponse pathway, more preferably those that activate the ATM (ataxiatelangiectasia, mutated) or ATR (ATM- and Rad3-related) protein kinases,or CHK1, or yet further CHK2 or p53. Examples of the latter includeionizing radiation, inhibitors of DNA replication, DNA polymeraseinhibitors and chromatic modifying agents or treatment including HDACinhibitors. Compositions that upregulate NKG2D ligands are furtherdescribed in Gasser et al (2005) Nature 436(7054):1186-90. NKG2D is anactivating receptor that interacts with the MHC class I-related MICA andMICB glycoproteins, among other ligands. MICA and MICB (Bauer et al.(1999) Science 285:727-729, the disclosure of which is incorporatedherein by reference) have no role in antigen presentation, are generallyonly found in intestinal epithelium, and can be stress-induced inpermissive types of cells by viral and bacterial infections, malignanttransformation, and proliferation. NKG2D is a C-type lectin-likeactivating receptor that signals through the associated DAP10 adaptorprotein, which is similar to CD28. It is expressed on most naturalkiller (NK) cells, NKT cells, γδ T cells CD8 T cells, and T cells, butnot, in general, on CD4 T cells. Other NKG2D ligands include ULBPproteins, e.g., ULBP-1, -2, and -3, originally identified as ligands forthe human cytomegalovirus glycoprotein UL16 (Cosman et al, (2001)Immunity 14: 123-133, the disclosure of which is incorporated herein byreference). Further NKG2D ligands include RAE1TG, a member of theULBP-like family of proteins (Bacon et al. (2004) J. Immunol.173:1078-1084).

Further anti-cancer agents include alkylating agents, cytotoxicantibiotics such as topoisomerase I inhibitors, topoisomerase IIinhibitors, plant derivatives, RNA/DNA antimetabolites, and antimitoticagents. Preferred examples may include, for example, cisplatin (CDDP),carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,mitomycin, etoposide (VP16), tamoxifen, raloxifene, taxol, gemcitabine,navelbine, transplatinum, 5-fluorouracil, vincristin, vinblastin andmethotrexate, or any analog or derivative variant of the foregoing.

Alkylating agents are substances that form compounds that are highlychemically reactive and rapidly form covalent bonds with suitablesubstances. One such target is DNA, not in its normal state but when thedouble helix has been unpaired by helicases. This exposes the ‘inside’of the DNA, which is susceptible to alkylation. Most alkylating agentsare bipolar, i.e., they contain two groups capable of reacting with DNA.They can thus form ‘bridges’ between two parts of a single strand of DNAor two separate strands; either way, this interferes with the actions ofthe enzymes involved with the replication process, which are unable tocomplete their effects. The cell then either dies because it isphysically unable to divide or because the abnormal DNA stimulatesapoptosis. Examples include nitrogen mustards (e.g. chlorambucil,cyclophosphamide), nitrosureas (e.g. carmustine, lomustine), metal salts(e.g. cisplatin, carboplatin, oxaliplatin), ethylenamine derivatives(e.g. thiotepa), alkyl sulphonates (e.g. busulphan) and triazenes (e.g.dacarbazine).

Antimetabolites are a group of chemicals that are similar in structureor function to naturally occurring metabolites required for thesynthesis of nucleic acids. Antimetabolite molecules mimic these normalmetabolites and either block the enzymes responsible for nucleic acidsynthesis or become incorporated into DNA, which produces an incorrectgenetic code and leads to apoptosis. There are three main classes ofantimetabolites. Folate is a substance that is necessary for thesynthesis of purine molecules. Folate analogues (e.g. methotrexate,raltritrexed) are similar to the folate molecule—substances such asmethotrexate can be used to inhibit the enzyme dihydrofolate reductase,resulting in insufficient production of the purine thymine. Pyrimidineanalogues (e.g. cytarabine, fluoroacil (5-FU), gemcitabine) resemblepyrimidine molecules and work by either inhibiting the synthesis ofnucleic acids (e.g. fluorouracil) or by becoming incorporated into DNA(e.g. cytarabine). Purine analogues (e.g. mercaptopurine, thioguanine,cladribine, fludarabine) work in similar ways to pyrimidine analogues,but may have additional (and ill-characterized) mechanisms of action.

Cytotoxic antibiotics are so called because they are all derived from anatural source, the Streptomyces group of bacteria. They affect thefunction and synthesis of nucleic acids in different ways. Theanthracycline group includes doxorubicin, daunorubicin and idarubicin.They intercalate with DNA and affect the topoisomerase II enzyme. ThisDNA gyrase splits the DNA double helix and reconnects it once torsionalforces have been relieved; the anthracyclines stabilize theDNA-topoisomerase II complex and thus prevent reconnection of thestrands. Dactinomycin and mitoxantrone have a similar mechanism ofaction. Bleomycin causes fragmentation of DNA chains. Mitomycinfunctions similar to the alkylating agents, causing DNA cross-linkage.

Plant derivatives include the vinca alkaloids such as vincristine andvinblastine bind to precursors of microtubules, preventing theirformation. This inhibits the process of mitosis. The taxanes (paclitaxeland docetaxel) also act on microtubules. They stabilize them in theirpolymerized state, which also causes the arrest of mitosis. Podophyllyumderivatives such as etoposide and teniposide are thought to inhibittopoisomerase II, while irinotecan and topotecan inhibit topoisomeraseI.

When infectious diseases are treated, the treatment may employ acomposition according to the invention, either alone or in combinationwith other treatments and/or therapeutic agents known for treating suchdiseases, including anti-viral agents, anti-fungal agents, antibacterialagents; antibiotics, anti-parasitic agents and anti-protozoal agents.When these methods involve additional treatments with additionaltherapeutic agents, those agents may be administered together with theantibodies of this invention as either a single dosage form or asseparate, multiple dosage forms. When administered as a separate dosageform, the additional agent may be administered prior to, simultaneouslywith, of following administration of the antibody of this invention.

The methods and antibodies of the present invention, particularlynon-depleting antibodies that block the interaction between MICA andNKG2D and/or inhibit MICA-induced NKG2D activity, can also be utilizedadvantageously for the treatment of inflammatory and autoimmunedisorders. Exemplary autoimmune or inflammatory conditions or disordersto be treated with the polypeptides, antibodies and other compounds ofthe invention, include, but are not limited to systemic lupuserythematosis, rheumatoid arthritis, juvenile chronic arthritis,psoriatic arthritis, osteoarthritis, spondyloarthropathies (ankylosingspondylitis), systemic sclerosis (scleroderma), idiopathic inflammatorymyopathies (dermatomyositis, polymyositis), Sjogren's syndrome,vasculitis, systemic vasculitis, temporal arteritis, atherosclerosis,sarcoidosis, myasthenia gravis, autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), pernicious anemia,autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura,immune-mediated thrombocytopenia), thyroiditis (Grave's disease,Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophicthyroiditis), diabetes mellitus, immune-mediated renal disease(glomerulonephritis, tubulointerstitial nephritis, autoimmuneoophiritis), autoimmune orchitis, autoimmune uveitis, anti-phospholipidsyndrome, demyelinating diseases of the central and peripheral nervoussystems such as multiple sclerosis, idiopathic demyelinatingpolyneuropathy or Guillain-Barre syndrome, and chronic inflammatorydemyelinating polyneuropathy, hepatobiliary diseases such as infectioushepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses),autoimmune chronic active hepatitis, viral hepatitis, primary biliarycirrhosis, granulomatous hepatitis, Wegener's granulomatosis, Behcet'sdisease, and sclerosing cholangitis, inflammatory bowel diseases such asulcerative colitis or Crohn's disease, celiac disease, gluten-sensitiveenteropathy, and Whipple's disease, autoimmune or immune-mediated skindiseases including bullous skin diseases, erythema multiforme andcontact dermatitis, dermitis herpetiformis, psoriasis, pemphigusvulgaris, vitiligo (leukoderma), allergic diseases such as asthma,allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, chronic obstructive pulmonary disease, and transplantationassociated diseases including graft rejection andgraft-versus-host-disease.

When inflammatory or autoimmune diseases are treated with anti-MICAantibody, the treatment methods this invention may further comprisetreating an individual with a second therapeutic agent, including agentsnormally utilized for the particular therapeutic purpose for which theantibody is being administered. The second therapeutic agent willnormally be administered in amounts typically used for that agent in amonotherapy for the particular disease or condition being treated. Inone embodiment, the second therapeutic agent is administered in a doseless than the generally accepted efficacious dose; for example, invarious embodiments, the composition comprises a dosage that is lessthan about 10% to 75% of the generally accepted efficacious dose isadministered. Preferably, the second therapeutic agent is an agent thatreduces proteolytic enzymes, an inflammatory mediator, or aproinflammatory cytokine such as TNF-α and/or interleukin-1 (IL-1).Preferably, the second therapeutic agent is DMARD or a DMD, optionallyfurther wherein the second therapeutic agent is methotrexate(Rheumatrex™, Trexall™), hydroxychloroquine (Plaquenil™), sulfasalazine(Azulfidine®), leflunomide (Arava™), a tumor necrosis factor inhibitor(e.g. a soluble TNFα receptor such as etanercept (Enbrel®), aneutralizing (preferably non-depleting) anti-TNFα antibody such asadalimumab (Humira™) or Certolizumab pegol (Cimzia™)), a T-cellcostimulatory blocking agent (e.g. abatacept (Orencia™)), aninterleukin-1 (IL-1) receptor antagonist therapy (anakinra (Kineret™)),an anti-BlyS antibody (Benlysta™), a proteosome inhibitor (e.g.bortezomib), a tyrosine kinase inhibitor, intramuscular gold, or anotherimmunomodulatory or cytotoxic, agent (e.g. azathioprine (Imuran™),cyclophosphamide, cyclosporine A (Neoral™, Sandimmune™)) or a kinaseinhibitor (e.g. a SYK kinase inhibitor such as fostimatinib (R788) or aJAK1, JAK2 inhibitors such as INCB28050, tanezumab or tasocitinib(CP-690,550)).

In the treatment methods of the invention, the antigen-binding compoundof the invention and the second therapeutic agent can be administeredseparately, together or sequentially, or in a cocktail. In someembodiments, the antigen-binding compound of the invention isadministered prior to the administration of the second therapeuticagent. For example, the antigen-binding compound of the invention can beadministered approximately 0 to 30 days prior to the administration ofthe second therapeutic agent. In some embodiments, an antigen-bindingcompound of the invention is administered from about 30 minutes to about2 weeks, from about 30 minutes to about 1 week, from about 1 hour toabout 2 hours, from about 2 hours to about 4 hours, from about 4 hoursto about 6 hours, from about 6 hours to about 8 hours, from about 8hours to 1 day, or from about 1 to 5 days prior to the administration ofthe second therapeutic agent. In some embodiments, an antigen-bindingcompound of the invention is administered concurrently with theadministration of the therapeutic agents. In some embodiments, anantigen-binding compound of the invention is administered after theadministration of the second therapeutic agent. For example, anantigen-binding compound of the invention can be administeredapproximately 0 to 30 days after the administration of the secondtherapeutic agent. In some embodiments, an antigen-binding compound ofthe invention is administered from about 30 minutes to about 2 weeks,from about 30 minutes to about 1 week, from about 1 hour to about 2hours, from about 2 hours to about 4 hours, from about 4 hours to about6 hours, from about 6 hours to about 8 hours, from about 8 hours to 1day; or from about 1 to 5 days after the administration of the secondtherapeutic agent.

The antigen-binding compounds of the invention can be included in kits.The kits may optionally further contain any number of antibodies and/orother compounds, e.g., 1, 2, 3, 4, or any other number of anti-MICAantibodies and/or other compounds. It will be appreciated that thisdescription of the contents of the kits is not limiting in any way. Forexample, the kit may contain other types of therapeutic or diagnosticagents. Preferably, the kits also include instructions for using theantibodies and/or agents, e.g., detailing the herein-described methods.

Pharmaceutical Formulations

Pharmaceutically acceptable carriers that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. The antibodies of this invention may be employed in a methodof modulating, e.g. inhibiting, the activity of MICA-expressing cells ina patient. This method comprises the step of contacting said compositionwith said patient. Such method will be useful for both prophylaxis andtherapeutic purposes.

For use in administration to a patient, the composition will beformulated for administration to the patient. The compositions of thepresent invention may be administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The used herein includes subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional and intracranialinjection or infusion techniques. The antibody can be present in asingle dose in an amount, for example, of between about 25 mg and 500mg.

Sterile injectable forms of the compositions of this invention may beaqueous or an oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or diglycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents that are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The compositions of this invention may be orally administered in anyorally acceptable dosage form including, but not limited to, capsules,tablets, aqueous suspensions or solutions. In the case of tablets fororal use, carriers commonly used include lactose and corn starch.Lubricating agents, such as magnesium stearate, are also typicallyadded. For oral administration in a capsule form, useful diluentsinclude, e.g., lactose. When aqueous suspensions are required for oraluse, the active ingredient is combined with emulsifying and suspendingagents. If desired, certain sweetening, flavoring or coloring agents mayalso be added.

Alternatively, the compositions of this invention may be administered inthe form of suppositories for rectal administration. These can beprepared by mixing the agent with a suitable non-irritating excipientthat is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum to release the drug. Such materialsinclude cocoa butter, beeswax and polyethylene glycols.

The compositions of this invention may also be administered topically,especially when the target of treatment includes areas or organs readilyaccessible by topical application, including diseases of the eye, theskin, or the lower intestinal tract. Suitable topical formulations arereadily prepared for each of these areas or organs. For topicalapplications, the compositions may be formulated in a suitable ointmentcontaining the active component suspended or dissolved in one or morecarriers. Carriers for topical administration of the compounds of thisinvention include, but are not limited to, mineral oil, liquidpetrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, thecompositions can be formulated in a suitable lotion or cream containingthe active components suspended or dissolved in one or morepharmaceutically acceptable carriers. Suitable carriers include, but arenot limited to, mineral oil, sorbitan monostearate, polysorbate 60,cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol andwater.

The present antibodies can be included in kits. The kits may optionallyfurther contain any number of antibodies and/or other compounds, e.g.,1, 2, 3, 4, or any other number of therapeutic antibodies and/orcompounds. It will be appreciated that this description of the contentsof the kits is not limiting in any way. For example, the kit may containother types of therapeutic compounds. Preferably, the kits also includeinstructions for using the antibodies, e.g., detailing theherein-described methods.

Dosage Forms

Therapeutic formulations of the antagonists used in accordance with thepresent invention are prepared for storage by mixing the antagonisthaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients, or stabilizers in the form oflyophilized formulations or aqueous solutions. For general informationconcerning formulations, see, e.g., Gilman et al. (eds.), ThePharmacological Bases of Therapeutics, 8^(th) Ed. (Pergamon Press,1990); Gennaro (ed.), Remington's Pharmaceutical Sciences, 18^(th)Edition (Mack Publishing Co., Easton, Pa., 1990); Avis et al. (eds.),Pharmaceutical Dosage Forms: Parenteral Medications (Dekker, New York,1993); Lieberman et al. (eds.), Pharmaceutical Dosage Forms: Tablets(Dekker, New York, 1990); Lieberman et al. (eds.) Pharmaceutical DosageForms: Disperse Systems (Dekker, New York, 1990); and Walters (ed.),Dermatological and Transdermal Formulations (Drugs and thePharmaceutical Sciences), Vol 119 (Dekker, New York, 2002).

Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low-molecular-weight (less than about 10 residues) polypeptides;proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilicpolymers such as polyvinylpyrrolidone; amino acids such as glycine,glutamine, asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as ethylenediaminetetraacetic acid(EDTA); sugars such as sucrose, mannitol, trehalose, or sorbitol;salt-forming counter-ions such as sodium; metal complexes (e.g.,Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™,PLURONICS™, or PEG.

Exemplary antibody formulations are described for instance in WO1998/56418, which describes a liquid multidose formulation for ananti-CD20 antibody, comprising 40 mg/mL rituximab, 25 mM acetate, 150 mMtrehalose, 0.9% benzyl alcohol, and 0.02% polysorbate20™ at pH 5.0 thathas a minimum shelf life of two years storage at 2-8° C. Anotheranti-CD20 formulation of interest comprises 10 mg/mL rituximab in 9.0mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mLpolysorbate80™, and Sterile Water for Injection, pH 6.5.

Lyophilized formulations adapted for subcutaneous administration aredescribed, for example, in U.S. Pat. No. 6,267,958 (Andya et al.). Suchlyophilized formulations may be reconstituted with a suitable diluent toa high protein concentration and the reconstituted formulation may beadministered subcutaneously to the mammal to be treated herein.

The formulation herein may also contain more than one active compound (asecond medicament as noted above), preferably those with complementaryactivities that do not adversely affect each other. The type andeffective amounts of such medicaments depend, for example, on the amountand type of B-cell antagonist present in the formulation, and clinicalparameters of the subjects. The preferred such second medicaments arenoted above.

The active ingredients may also be entrapped in microcapsules prepared,e.g., by coacervation techniques or by interfacial polymerization, forexample, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles, and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra, for example.

Sustained-release formulations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antagonist, which matrices are inthe form of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andγethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes. Pharmaceutically acceptable carriers that may be used inthese compositions include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Further aspects and advantages of this invention will be disclosed inthe following experimental section, which should be regarded asillustrative and not limiting the scope of this application.

EXAMPLES Example 1: Generation of Anti-MICA Antibodies

Immunization #1

To obtain anti-human MICA antibodies, Balb/c mice were immunized with arecombinant human MICA extracellular domain recombinant-Fc protein(MICA*019 allele, available from R&D Systems). Mice received oneprimo-immunization with an emulsion of 50 μg MICA protein and CompleteFreund Adjuvant, intraperitoneally, a 2^(nd) immunization with anemulsion of 50 μg MICA protein and Incomplete Freund Adjuvant,intraperitoneally, and finally a boost with 10 μg MICA protein,intravenously. Immune spleen cells were fused 3 days after the boostwith X63.Ag8.653 immortalized B cells, and cultured in the presence ofirradiated spleen cells.

Primary screen: Supernatant (SN) of growing clones were tested in aprimary screen by flow cytometry using Baf/3 cell line transfected witha MICA*019 construct. Positive supernatants were selected and tested forlack of binding by flow cytometry to untransfected Baf/3 cell line.Briefly, for FACS screening, the presence of reacting antibodies insupernatants was revealed by Goat anti-mouse polyclonal antibody (pAb)labeled with PE.

Secondary screen: Supernatants of the clones were also tested using anELISA assay to assess the capacity to block the interaction between MICAextracellular domain recombinant-Fc protein (R&D Systems) and NKG2Dextracellular domain recombinant-Fc protein. Potentially interestinghybridomas selected from the initial screening were cloned by limitingdilution techniques in 96-wells plates. The resulting antibodiessupernatant 9C10 of IgG2b isotype and 6E4, 20C6 and 19E9 of IgG1 isotypewere obtained.

Immunization #2

To obtain anti-human MICA antibodies, Balb/c mice were immunized with arecombinant human MICA extracellular domain recombinant-His protein(MICA*001 allele). Mice received one primo-immunization with an emulsionof 50 μg MICA protein and Complete Freund Adjuvant, intraperitoneally, a2^(nd) immunization with an emulsion of 50 μg MICA protein andIncomplete Freund Adjuvant, intraperitoneally, and one boost with 10 μgMICA protein, intravenously. Immune spleen cells were fused withX63.Ag8.653 immortalized B cells, and cultured in the presence ofirradiated spleen cells.

Primary screen: Supernatant (SN) of growing clones were tested in aprimary screen by flow cytometry using a mixture of cells of differentC1R-based cell lines, wherein each cell line was transfected with adifferent construct (either MICA*001, MICA*004, MICA*007 or MICA*008).Briefly, for FACS screening, the presence of reacting antibodies insupernatants was revealed by Goat anti-mouse polyclonal antibody (pAb)labeled with PE.

Secondary screen: Supernatants of the clones were also tested using anELISA assay to assess the capacity to bind MICA extracellular domain α3recombinant protein without binding to recombinant human MICA fullextracellular domain recombinant-His protein (MICA*001 allele).Potentially interesting hybridomas selected from the initial screeningwere cloned by limiting dilution techniques in 96-wells plates.

MICA extracellular domain α3 antibodies were obtained, including clone16A8 of IgG2a isotype.

Immunization #3

To obtain anti-human MICA antibodies, Balb/c mice were immunized with amix of 10 millions C1R cells transfected with MICA*01, MICA*04, MICA*07or MICA*08 at a 1:1:1:1 ratio (2.5 millions of each transfectant cells).Mice received one primo-immunization with a total of 10 millionstransfectant cells and Complete Freund Adjuvant, intraperitoneally, a2^(nd) immunization with 10 millions transfectants cells (2.5 millionsof each transfectant cells) and incomplete Freund Adjuvant,intraperitoneally, and one boost with 1 million transfectant cells (0.25million of each transfectant cells), intravenously. Immune spleen cellswere fused with X63.Ag8.653 immortalized B cells, and cultured in thepresence of irradiated spleen cells.

Primary screen: Supernatant (SN) of growing clones were tested in aprimary screen by flow cytometry using a mixture of cells of differentC1R-based cell lines and Baf/3 cell line, wherein each cell line wastransfected with a different construct (either MICA*001, MICA*004,MICA*007 or MICA*008 for C1R and MICA*019 for Baf/3). Briefly, for FACSscreening, the presence of reacting antibodies in supernatants wasrevealed by Goat anti-mouse polyclonal antibody (pAb) labeled with PE.

Secondary screen: Supernatants of the clones were also tested using anELISA assay to assess the capacity to bind MICA extracellular domain α3recombinant protein without binding to recombinant human MICA fullextracellular domain recombinant-Fc protein (MICA*019 allele).Potentially interesting hybridomas selected from the initial screeningwere cloned by limiting dilution techniques in 96-wells plates.

The resulting antibodies supernatant 12A10, 10A7, 18E8, 10F3 and 15F9 ofIgG1 isotype and 14B4 of IgM isotype were obtained.

Antibodies 9C10, 19E9, 6E4, 20C6, 16A8, 12A10, 10A7, 18E8, 10F3, 15F9and 14B4 were subsequently chimerized to human IgG1 isotype (alsoreferred to as CHG1-M-MIA-9C10, CHG1-M-MIA-19E9, CHG1-M-MIA-6E4,CHG1-M-MIA-20C6, CHG1-M-MIA-16A8, CHG1-M-MIA-12A10, CHG1-M-MIA-10A7,CHG1-M-MIA-18E8, CHG1-M-MIA-10F3, CHG1-M-MIA-15F9 and CHG1-M-MIA-14B4respectively).

Example 2—Binding to Immobilized MICA Proteins

The binding of 6E4, 20C6 and 16A8 to either recombinant human MICAextracellular domain recombinant protein monomers or dimers (MICA*019allele-Fc protein or MICA*001-His protein) as well as other NKG2Dligands MICB and ULBP1-2 was analyzed by Surface Plasmon Resonance (SPR)using a Biacore T100 apparatus to obtain monovalent and bivalentaffinity, respectively.

For protein immobilization, recombinant proteins: MICA-Fc MICB-Fc (R&DSystems 1599-MB, Accession no. CAI18747, SEQ ID NO: 6), ULBP1-Fc,ULBP2-Fc were immobilized covalently to carboxyl groups in the dextranlayer on a Sensor Chip CM5 (chip). The chip surface was activated withEDC/NHS (N-ethyl-N′-(3-dimethylaminopropyl) carbodiimidehydrochlorideand N-hydroxysuccinimide (Biacore GE Healthcare)). Proteins were dilutedto 10 μg/ml in coupling buffer (10 mM acetate, pH 5.6) and injecteduntil the appropriate immobilization level was reached (i.e. 1000 to1600 RU for binding study). Deactivation of the remaining activatedgroups was performed using 100 mM ethanolamine pH 8 (Biacore GEHealthcare).

Antibody binding analysis was run using HBS-EP+(neutral pH). Theanti-MICA antibodies (diluted at a concentration of 10 μg/ml in therunning buffer), were injected for 2 min at a constant flow rate of 10μl/min on dextran layers containing immobilized recombinant targetproteins and allowed to dissociate for 2 min before regeneration by aten second injection of 10 mM NaOH, 500 mM NaCl regeneration buffer at aflow rate of 40 μl/min.

For bivalent affinity measurement, MICA-His protein was immobilized viaNi²⁺/NTA chelation on a Sensor Chip NTA (carboxymethylated dextranpre-immobilized with nitrilotriacetic acid (NTA)). Anti-MICA antibodieswere diluted to a concentration series (0.01 to 100 nM) in the runningbuffer HBS-P and injected over the immobilized antigen.

Each cycle consisted of three steps. Firstly, the NTA chip was activatedwith NiSO₄ (500 mM). Secondly, the MICA-His protein (diluted to 10 μg/mlin running buffer HBS-P) was immobilized onto the surface via a 2 mininjection at a constant flowrate of 10 μL/min. Then the antibody wasinjected over the immobilized antigen for 2 min at the flow rate of 40μl/min. Subsequently, the running buffer was injected for 5 min at 40μl/min for antibody dissociation analysis. After each cycle, the surfacewas regenerated by a 60s injection of 0.5M EDTA, pH 8.3 to completelystrip the surface of the remaining antigen and antibody. The resultingsensorgrams were analyzed by global fitting using the appropriate model.

For monovalent affinity measurement, chimeric anti-MICA antibodies werecaptured onto a Protein-G chip.

Affinities were determined using single cycle kinetics (SCK). SCK cycleswere as follows: Successive 120s injections at 30 μl/min of 5 serialdilutions (0.01 to 100 nM) of MICA-His or MICA-BirA in ascending order(or of buffer for the blank injections to be subtracted during theanalysis). After the last concentration is injected, complex is left todissociate for 600s before regeneration by a 10s injection ofappropriate regeneration buffer at 40 μl/min. After the regeneration,chip is left to stabilise for 60s in running buffer. Curves are fittedusing Biacore T100 Evaluation software.

The bivalent mean K_(D) (M) at pH 7.4 for MICA binding forbiotin-conjugated mouse antibody 6E4 (on MICA*001-His) was 3.099⁻¹¹ M),while the monovalent affinity was 2.0*10⁻⁹ M. The bivalent mean K_(D)(M) at pH 7.4 for biotin-conjugated mouse antibody 20C6 (onMICA*001-His) was 3.3*10⁻¹⁰ M, while the monovalent affinity was6.5*10⁻⁹ M.

The bivalent mean K_(D) (M) at pH 7.4 for MICA binding forbiotin-conjugated mouse antibody 9C10 (on MICA*001-His) was 6.2*10⁻¹³ M.The bivalent mean K_(D) (M) at pH 7.4 for MICA binding forbiotin-conjugated mouse antibody 19E9 (on MICA*001-His) was 3.2*10⁻¹³ M,while the monovalent affinity was 7.8*10⁻¹⁰ M.

The monovalent affinity (mean KD) at pH7.4 on MICA-BirA forCHG1-M-MIA-19E9 antibody was 6.5*10⁻⁹ M (n=3) The monovalent affinity(mean KD) at pH7.4 on MICA-BirA for CHG1-M-MIA-20C6 antibody was 4*10⁻⁸M (n=3)

The monovalent affinity (mean KD) at pH7.4 on MICA-BirA forCHG1-M-MIA-6E4 antibody was 8.9*10⁻⁹ M (n=4)

The monovalent affinity (mean KD) at pH7.4 on MICA-BirA forCHG1-M-MIA2-16A8 antibody was 1.15*10⁻⁸ M (n=4)

The monovalent affinity (mean KD) at pH7.4 on MICA-BirA forCHG1-M-MIA-15F9 antibody was 6.1*10⁻⁸ M (n=1)

The monovalent affinity (mean KD) at pH7.4 on MICA-BirA forCHG1-M-MIA-12A10 antibody was 5.3*10⁻⁸ M (n=1) The monovalent affinity(mean KD) at pH7.4 on MICA-BirA for CHG1-M-MIA-18E8 antibody was5.4*10⁻⁸ M (n=1)

The monovalent affinity (mean KD) at pH7.4 on MICA-BirA forCHG1-M-MIA-10F3 antibody was 8.3*10⁻⁹ M (n=1)

Example 3—Binding to MICA Alleles

The binding of antibodies obtained from the first, second and thirdimmunization series (including CHG1-M-MIA-19E9, CHG1-M-MIA-9C10,CHG1-M-MIA-6E4, CHG1-M-MIA-20C6, CHG1-M-MIA-16A8, CHG1-M-MIA-12A10,CHG1-M-MIA-10A7, CHG1-M-MIA-18E8, CHG1-M-MIA-10F3, CHG1-M-MIA-15F9 andCHG1-M-MIA-14B4) were tested for binding to MICA-expressing CR1transfectant cells C1R-MICA*001, C1R-MICA*004, C1R-MICA*007 andC1R-MICA*008 (Pr. A. Steinle, Eberhard-Karls University Tuebingen,Germany) described in Salih et al. (2003) Blood 102(4): 1389-91396.Binding was analyzed by flow cytometry.

Flow Cytometry.

Cells were harvested and stained in PBS 1×/BSA 0.2%/EDTA 2 mM bufferduring 30 minutes at 4° C. using a dose-range of the anti-MICA mAbs.After two washes in staining buffer, cells were stained for 30 min at 4°C. with mouse anti-human IgG1-PE monoclonal antibodies (1/11). After twowashes, stainings were acquired on a BD FACS Canto II and analyzed usingthe FlowJo software.

Results.

Antibodies CHG1-M-MIA-19E9, CHG1-M-MIA-9C10, CHG1-M-MIA-6E4,CHG1-M-MIA-20C6, CHG1-M-MIA-12A10, CHG1-M-MIA-10A7, CHG1-M-MIA-18E8,CHG1-M-MIA-10F3, and CHG1-M-MIA-15F9 bound to each of the C1R-MICA*001,C1R-MICA*004, C1R-MICA*007 and C1R-MICA*008 cells (see FIG. 1A forantibodies CHG1-M-MIA-19E9, CHG1-M-MIA-9C10, CHG1-M-MIA-6E4,CHG1-M-MIA-20C6, CHG1-M-MIA-12A10, CHG1-M-MIA-10F3, and FIG. 1B forantibodies, CHG1-M-MIA-18E8, CHG1-M-MIA-15F9, CHG1-M-MIA-10A7). Howeverother antibodies tested were allele specific and did not recognize allof MICA*001,*004, *007 and *008. As an example of antibodies that do notrecognize all of tested MICA alleles, two anti-alpha3 domain antibodies,CHG1-M-MIA-16A8 and CHG1-M-MIA-14B4 are shown in FIG. 1B. EC50 valuesare shown in Table C in μg/ml (calculated using a 4-parameter logisticfit).

TABLE C EC50 values in μg/ml of indicated chimeric anti-MICA antibodieson C1R transfectant cells C1R- C1R- C1R- C1R- MICA*01 MICA*04 MICA*07MICA*08 CHG1-M-MIA-9C10 2.09 0.13 2.83 0.13 CHG1-M-MIA-12A10 1.30 0.261.43 0.25 CHG1-M-MIA-19E9 1.40 0.04 0.23 0.07 CHG1-M-MIA-18E8 0.50 0.150.30 0.24 CHG1-M-MIA-10F3 0.47 0.17 0.94 0.26 CHG1-M-MIA-15F9 0.68 0.431.81 0.79 CHG1-M-MIA-6E4 1.96 2.67 1.23 CHG1-M-MIA-20C6 0.26 0.05 0.400.13 CHG1-M-MIA-10A7 2.03 0.34 0.97 0.77 CHG1-M-MIA-16A8 1.07 0.44CHG1-M-MIA-14B4 12.7 0.24 0.30

Example 4—Epitope Mapping

Antibodies were further tested for binding to various MICA*001 mutants.MICA mutants were generated by PCR (see Table D below). All the Mx-Rprimers were used with the following 5′ primerTACGACTCACAAGCTTACCGCCACCATGGGGCT GGGACCGGTCTTC (SEQ ID NO: 135). Allthe Mx-F primers were used with the following 3′ primerCCGCCCCGACTCTAGATTACTAGGCGCCCTCAGTGGAGC (SEQ ID NO: 136). The sequencesamplified were run on agarose gel then purified using the Macherey NagelPCR Clean-Up Gel Extraction kit (reference 740609). To create mutant 3,it was necessary to do a third PCR. Primers used for these PCR wereM3a-F primer (5′-ACGGTGCTGTCCGCGGATGGATCTGTGCAGTCAG-3′) (SEQ ID NO: 137)with the M3b-R primer (5′-CCTGCTTTCTGGTCCTTGATATGAGCCAGGGTC-3′) (SEQ IDNO: 138). The two or three PCR products generated for each mutant werethen ligated into a SELEXIS pSUREtech 192 (HYGRO) vector, digested withthe restriction enzyme HindIII and XBaI, with the ClonTech InFusionsystem (reference 639644) according to the manufacturer's instructions.

After sequencing, the vectors containing the mutated sequences wereprepared as Miniprep using the Promega PureYield™ Plasmid MiniprepSystem (reference A1222). Vectors were then used for CHOK1SV celltransfection using OZ BIOSCIENCES's HYPE-5™ Transfection Kit (referenceHYR10003) according to the manufacturer instructions. Transfection wereperformed using 300 ng of DNA at 1:8 ratio HYPE-5:DNA.

TABLE DMICA*001 mutant list with targeted amino acid and primers sequences used to generate mutantMutants Reverse primers Forward primers Number 1 M1-R M1-F R6A + N8A5′-AGGGCATAAGCAAGACTGTGGGGCTCAGC 5′-TCTTGCTTATGCCCTCACGGTGCTGTCCTGAGCAG-3′ (SEQ ID NO: 139) GGATG-3′ (SEQ ID NO: 140) Number 2 M2-R M2-FL12A + Q19A 5′-ACGCCACAGATCCATCCCAGGACGCCACC5′-ATGGATCTGTGGCGTCAGGGTTTCTCACTG GTGAG-3′ (SEQ ID NO: 141)AGG-3′ (SEQ ID NO: 142) Number 3 M3a-R M3b-F W14A + E85A5′-CGCGGACAGCACCGTGAGGTTATAACGAA 5′-GGACCAGAAAGCAGGCTTGCATTCCCTACAGACTGTG-3′ (SEQ ID NO: 143) GGAG-3′ (SEQ ID NO: 144) Number 4 M4-R M4-FD15A + S17A 5′-CTCCAGCCCAGGACAGCACCGTGAGGTTA5′-TGTCCTGGGCTGGAGCTGTGCAGTCAGGGT TAACGA-3′ (SEQ ID NO: 145)TTCTC-3′ (SEQ ID NO: 146) Number 5 M5-R M5-F S20A5′-AGTGAGAAACCCTGCCTGCACAGATCCAT 5′-GCAGGGTTTCTCACTGAGGTACATCTGGATCCCAGGACA-3′ (SEQ ID NO: 147) GGTCA-3′ (SEQ ID NO: 148) Number 6 M6-RM6-F E25A + H27A + 5′-CCAGAGCTACCGCAGTGAGAAACCCTGAC5′-CTGCGGTAGCTCTGGATGGTCAGGCCTTCC P32A TGCAC-3′ (SEQ ID NO: 149)TGCG-3′ (SEQ ID NO: 150) Number 7 M7-R M7-F R35A + K44A5′-TGCCTGTCACAGGCCAGGAAGGGCTGACC 5′-GGCCTGTGACAGGCAGAAATGCAGGGCAGCATCCAGA-3′ (SEQ ID NO: 151) GCCCCAGGGA-3′ (SEQ ID NO: 152) Number 8 M8-RM8-F D37A + R38S + 5′-CATTTCGCACTGGCACAGCGCAGGAAGGG5′-TGCCAGTGCGAAATGCAGGGCAAAGCCCCA Q39A CTGACCATC-3′ (SEQ ID NO: 153)GGGACAG-3′ (SEQ ID NO: 154) Number 9 M9-R M9-F K40A + R42A5′-CCGCGCATGCCTGCCTGTCACAGCGCAGG 5′-GGCAGGCATGCGCGGCAAAGCCCCAGGGACAAGG-3′ (SEQ ID NO: 155) AGTG-3′ (SEQ ID NO: 156) Number 10 M10-R M10-FQ48A + W49S 5′-TTCTGCCGACGCTCCCTGGGGCTTTGCTC5′-GGAGCGTCGGCAGAAGATGTCCTGGGAAAT TGCATTTC-3′ (SEQ ID NO: 157)AAGAC-3′ (SEQ ID NO: 158) Number 11 M11-R M11-F E51S + D52A +5′-CGCGGAAGCTGATGCCCACTGTCCCTGAG 5′-GCATCAGCTTCCGCGGGAAATAAGACATGGV53S + L54A GCTTTG-3′ (SEQ ID NO: 159) GACAG-3′ (SEQ ID NO: 160)Number 12 M12-R M12-F N56A + K57S + 5′-TCCCATGCACTAGCTCCCAGGACATCTTC5′-AGCTAGTGCATGGGACAGAGAGACCAGAGA T58A TGCCCAC-3′ (SEQ ID NO: 161)CTTGAC-3′ (SEQ ID NO: 162) Number 13 M13-R M13-F R61A + R64A5′-CTGCGGTCTCTGCGTcccatgtcttattt 5′-ACGCAGAGACCGCAGACTTGACAGGGAACGcccaggacatc-3′ (SEQ ID NO: 163) GAAAGGAC-3′ (SEQ ID NO: 164) Number 14M14-R M14-F K71A 5′-CTGAGGTCCGCTCCGTTCCCTGTCAAGTC5′-CGGAGCGGACCTCAGGATGACCCTGGCTCA TCTG-3′ (SEQ ID NO: 165)TATC-3′ (SEQ ID NO: 166) Number 15 M15-R M15-F R74A + M75S +5′-AGCAGACAGGGTACTCGCGAGGTCCTTTC 5′-AGTACCCTGTCTGCTATCAAGGACCAGAAAA78S + H79A CGTTCCCTG-3′ (SEQ ID NO: 167) GAAGG-3′ (SEQ ID NO: 168)Number 16 M16-R M16-F K81A + D82A 5′-TTCTTTCTGGGCCGCGATATGAGCCAGGG5′-GCGGCCCAGAAAGAAGGCTTGCATTCCCTC TCATC-3′ (SEQ ID NO: 169)CAG-3′ (SEQ ID NO: 170) Number 17 M17-R M17-F Q83A + K84A5′-TGCCGCGTCCTTGATATGAGCCAGGGTCA 5′-ATCAAGGACGCGGCAGAAGGCTTGCATTCCTCCTGAG-3′ (SEQ ID NO: 171) CTCCAG-3′ (SEQ ID NO: 172) Number 18 M18-RM18-F E97A + H99A 5′-TCTTCAGCGATCGCACAGACCCTAATCTC5′-TGCGATCGCTGAAGACAACAGCACCAGGAG CTGGAGG-3′ (SEQ ID NO: 173)TTCCCAGC-3′ (SEQ ID NO: 174) Number 19 M19-R M19-F E100A + D101S +5′-TGGCGGATGCATGGATCTCACAGACCCTA 5′-TCCATGCATCCGCCAGCACCAGGAGCTCCC N102AATCTCC-3′ (SEQ ID NO: 175) AGCATTTC-3′ (SEQ ID NO: 176) Number 20 M20-RM20-F S103A + T104S + 5′-CTCGCGGAGGCGTTGTCTTCATGGATCTC5′-CAACGCCTCCGCGAGCTCCCAGCATTTCTA R105A ACAGACC-3′ (SEQ ID NO: 177)CTACG-3′ (SEQ ID NO: 178) Number 21 M21-R M21-F H109A + Y111A +5′-GGCGAAAGCCTGGGAGCTCCTGGTGCTGT 5′-TCCCAGGCTTTCGCCTACGATGGCGAGGCC L116ATGTCTTCATGGATCTCAC-3′ TTCCTCTCCCAAAACC-3′ (SEQ ID NO: 179)(SEQ ID NO: 180) Number 22 M22-R M22-F D113A + E115A5′-GCCCCAGCGTAGTAGAAATGCTGGGAGCT 5′-CTACTACGCTGGGGCGCTCTTCCTCTCCCACCTGGTGC-3′ (SEQ ID NO: 181) AAACCTG-3′ (SEQ ID NO: 182) Number 23 M23-RM23-F N121A + E123S 5′-TCGACAGGGCTTGGGAGAGGAAGAGCTCC5′-CCCAAGCCCTGTCGACTAAGGAATGGACAA CCATCG-3′ (SEQ ID NO: 183)TGCC-3′ (SEQ ID NO: 184) Number 24 M24-R M24-F T124A + E126A5′-TCCATGCCTTAGCCTCCAGGTTTTGGGAG 5′-AGGCTAAGGCATGGACAATGCCCCAGTCCTAGGAAG-3′ (SEQ ID NO: 185) CCAG-3′ (SEQ ID NO: 186) Number 25 M25-RM25-F T128A + M129S + 5′-GACTGGGCCGATGCCCATTCCTTAGTCTC5′-GGCATCGGCCCAGTCCTCCAGAGCTCAGAC P130A CAGGTTTTG-3′ (SEQ ID NO: 187)CTTG-3′ (SEQ ID NO: 188) Number 26 M26-R M26-F Q131A + S132A +5′-GGAGGCCGCGGGCATTGTCCATTCCTTAG 5′-ATGCCCGCGGCCTCCAGAGCTTCGACCTTG Q136STCTCCAG-3′ (SEQ ID NO: 189) GCCATGAAC-3′ (SEQ ID NO: 190) Number 27M27-R M27-F S133A + R134S + 5′-GCCTGAGCGCTGGCGGATTGTGGCATTGT5′-CGCCAGCGCTCAGGCCTTGGCCATGAACGT T137A CCATTCCTTAGTCTCCAG-3′CAGG-3′ (SEQ ID NO: 192) (SEQ ID NO: 191) Number 28 M28-R M28-FM140S + N141A + 5′-GCGGAGGCCGAGGCCAAGGTCTGAGCTCT5′-GGCCTCGGCCTCCGCGAATTTCTTGAAGGA R143S + N144AGGAGG-3′ (SEQ ID NO: 193) AGATGCC-3′ (SEQ ID NO: 194) Number 29 M29-RM29-F K147S + E148A + 5′-CAGATGCCGACAAGAAATTCCTGACGTTC5′-TCTTGTCGGCATCTGCCATGAAGACCAAGA D149S ATGG-3′ (SEQ ID NO: 195)CACAC-3′ (SEQ ID NO: 196) Number 30 M30-R M30-F A150S + M151A +5′-TGTCTTGGTCTTCGCGGAATCTTCCTTCA 5′-GCGAAGACCAAGACAGCCTATCACGCTATG H156AAGAAATTCCTG-3′ (SEQ ID NO: 197) CATGCAG-3′ (SEQ ID NO: 198) Number 31M31-R M31-F T153A + K154S + 5′-ATAGTGTGCCGAGGCCTTCATGGCATCTT5′-GCCTCGGCACACTATCACGCTATGCATGCA T155A CCTTC-3′ (SEQ ID NO: 199)GAC-3′ (SEQ ID NO: 200) Number 32 M32-R M32-F H158A + H161S5′-CAGACATAGCGGCATAGTGTGTCTTGGTC 5′-ATGCCGCTATGTCTGCAGACTGCCTGCAGGTTCATGG-3′ (SEQ ID NO: 201) AACTAC-3′ (SEQ ID NO: 202) Number 33 M33-RM33-F A162S + D163A + 5′-CGCCAGGCAGGCTGAATGCATAGCGTGAT5′-TCAGCCTGCCTGGCGGAACTACGGCGATAT Q166A AGTGTGTC-3′ (SEQ ID NO: 203)CTAAAATCC-3′ (SEQ ID NO: 204) Number 34 M34-R M34-F E167A + R169S +5′-TATGCCGATAGTGCCTGCAGGCAGTCTGC 5′-GGCACTATCGGCATATCTAAAATCCGGCGT R170AATGCATAG-3′ (SEQ ID NO: 205) AGTCCTG-3′ (SEQ ID NO: 206) Number 35 M35-RM35-F L172A + K173S + 5′-TACGCCGGCTGATGCATATCGCCGTAGTT5′-GCATCAGCCGGCGTAGTCCTGAGGAGAACA S174A CCTGC-3′ (SEQ ID NO: 207)GTGC-3′ (SEQ ID NO: 208) Number 36 M36-R M36-F L178A + R179S +5′-GCGCTCGCGACTACGCCGGATTTTAGATA 5′-CGTAGTCGCGAGCGCAACAGTGCCTCCCAT R180ATCGCCGTAG-3′ (SEQ ID NO: 209) GGTGAATGTC-3′ (SEQ ID NO: 210) Number 37M37-R M37-F P183A + P184A 5′-TTCACCATGGCGGCCACTGTTCTCCTCAG5′-GGCCGCCATGGTGAATGTCACCCGCAGCGA GACTACGC-3′ (SEQ ID NO: 211)GGCCTCAG-3′ (SEQ ID NO: 212) Number 38 M38-R M38-F M185A + V186S +5′-CAGCCGACGCGGGGGGCACTGTTCTCCTC 5′-CCCCCGCGTCGGCTGTCACCCGCAGCGAGG N187AAGGACTAC-3′ (SEQ ID NO: 213) CCTCAGAG-3′ (SEQ ID NO: 214) Number 39M39-R M39-F R190S + S191A + 5′-CCGAGGCGCTGGTGACATTCACCATGGGG5′-TCACCAGCGCCTCGGCCTCAGAGGGCAACA E192S + A193AGGCACTGTTC-3′ (SEQ ID NO: 215) TTACC-3′ (SEQ ID NO: 216) Number 40 M40-RM40-F N197A 5′-ATGGCGCCCGATGCGGCCTCGCTGCGGGT5′-CGCATCGGGCGCCATTACCGTGACATGCAG GACATTC-3′ (SEQ ID NO: 217)GGCTTC-3′ (SEQ ID NO: 218) Number 41 M41-R M41-F R203S + S205A +5′-GCTCAAAGATACCCCATCCTGACGCCAGC 5′-GGGGTATCTTTGAGCCACGACACCCAGCAG Q242ATCAGTGTGATATTCCAGGGATAGAAGCCA TGGGGGGATGTCCTGCCTGATGGGAATGGAGCAGCACTGCATGTCACGGTAATG-3′ ACCTACGCGACCTGGGTGGCCACCAG-3′(SEQ ID NO: 219) (SEQ ID NO: 220) Number 42 M42-R M42-F W210A + N211S +5′-CAGTGTGGCACTCGCGGGATAGAAGCCAG 5′-GCGAGTGCCACACTGAGCTGGCGTCAGGAT I212AAAGC-3′ (SEQ ID NO: 221) GG-3′ (SEQ ID NO: 222) Number 43 M43-R M43-FR217A + V221A 5′-AGATGCCCCATCCTGAGCCCAGCTCAGTG5′-CAGGATGGGGCATCTTTGAGCCACGACACC TGATATTCCAG-3′ (SEQ ID NO: 223)CAGCAG-3′ (SEQ ID NO: 224) Number 44 M44-R M44-F Q218A + D219A +5′-TCCCATCAGGCAGGACATCCCCCCACTGC 5′-TCCTGCCTGATGGGAATGGAACCTACCAGA R256ATGGGTGTCGTGGCTCAAAGATACCCCAGC CCTGGGTGGCCACCAGGATTTGCCAAGGAGCGCACGCCAGCTCAGTGTG-3′ AGGAGCAGGCGTTCACCTGCTACATG-3′ (SEQ ID NO: 225)(SEQ ID NO: 226) Number 45 M45-R M45-F S224A + H225S +5′-TGGGTGGCGCTGGCCAAAGATACCCCATC 5′-GGCCAGCGCCACCCAGCAGTGGGGGGATGT D226ACTGAC-3′ (SEQ ID NO: 227) CCTG-3′ (SEQ ID NO: 228) Number 46 M46-R M46-FT227A + Q228S + 5′-CCCCCACGCCGAGGCGTCGTGGCTCAAAG5′-GCCTCGGCGTGGGGGGATGTCCTGCCTGAT Q229A ATACC-3′ (SEQ ID NO: 229)GGGAATG-3′ (SEQ ID NO: 230) Number 47 M47-R M47-F W230A + D232A5′-GCCCCCGCCTGCTGGGTGTCGTGGCTCAA 5′-CCAGCAGGCGGGGGCTGTCCTGCCTGATGGAGATACC-3′ (SEQ ID NO: 231) GAATGG-3′ (SEQ ID NO: 232) Number 48 M48-RM48-F L234A + P235S + 5′-TCCCAGCAGACGCGACATCCCCCCACTGC5′-TCGCGTCTGCTGGGAATGGAACCTACCAGA D236A TGGGTGTC-3′ (SEQ ID NO: 233)CCTGGGTG-3′ (SEQ ID NO: 234) Number 49 M49-R M49-F N238A + Y241A5′-CAGGTCTGGGCGGTTCCAGCCCCATCAGG 5′-AACCGCCCAGACCTGGGTTGCCACCAGGATCAGGACA-3′ (SEQ ID NO: 235) TTGCCAAG-3′ (SEQ ID NO: 236) Number 50 M50-RM50-F W244A 5′-GTGGCCACCGCGGTCTGGTAGGTTCCATT5′-GACCGCGGTGGCCACCAGGATTTGCCAAGG CCCATC-3′ (SEQ ID NO: 237)AGAGGAG-3′ (SEQ ID NO: 238) Number 51 M51-R M51-F R248A5′-GGCAAATCGCGGTGGCCACCCAGGTCTGG 5′-CCACCGCGATTTGCCAAGGAGAGGAGCAGATAGGTTC-3′ (SEQ ID NO: 239) GGTTCAC-3′ (SEQ ID NO: 240) Number 52 M52-RM52-F C250A + E253A 5′-CCGCTCCTTGGGCAATCCTGGTGGCCACC5′-TTGCCCAAGGAGCGGAGCAGAGGTTCACCT CAGGTC-3′ (SEQ ID NO: 241)GCTAC-3′ (SEQ ID NO: 242) Number 53 M53-R M53-F Q251A + E254A5′-GCCTCTCCTGCGCAAATCCTGGTGGCCAC 5′-TTGCGCAGGAGAGGCGCAGAGGTTCACCTGCCAGGTC-3′ (SEQ ID NO: 243) CTAC-3′ (SEQ ID NO: 244) Number 54 M54-RM54-F Q255A 5′-GCCTCCTCTCCTTGGCAAATCCTGGTGGC5′-CCAAGGAGAGGAGGCGAGGTTCACCTGCTA CACCCAG-3′ (SEQ ID NO: 245)CATGG-3′ (SEQ ID NO: 246) Number 55 M55-R M55-F T258A + T269A +5′-TTCCCGCTGTGTTCCATGTAGCAGGCGAA 5′-GGAACACAGCGGGAATCACAGCGCTCACGC P271ACCTCTGCTCCTC-3′ (SEQ ID NO: 247) TGTGCCCTCTGGGAAAG-3′ (SEQ ID NO: 248)Number 56 M56-R M56-F Y260A + E262S + 5′-GCTGTGTGACATGGCGCAGGTGAACCTCT5′-GCCATGTCACACAGCGGGAATGCCAGCACT H267A GCTCCTC-3′ (SEQ ID NO: 249)CACCCTGTGC-3′ (SEQ ID NO: 250) Number 57 M57-R M57-F N266A5′-TGTGAGCCCCGCTGTGTTCCATGTAGCAG 5′-ACAGCGGGGCTCACAGCACTCACCCTGTGCGTGAACCTC-3′ (SEQ ID NO: 251) CCTCTG-3′ (SEQ ID NO: 252) Number 58 M3a-RM3a-F W14A 5′-CGCGGACAGCACCGTGAGGTTATAACGAA5′-ACGGTGCTGTCCGCGGATGGATCTGTGCAG GACTGTG-3′ (SEQ ID NO: 253)TCAG-3′ (SEQ ID NO: 254) Number 59 M3b-R M3b-F E85A5′-CCTGCTTTCTGGTCCTTGATATGAGCCAG 5′-GGACCAGAAAGCAGGCTTGCATTCCCTACAGGTC-3′ (SEQ ID NO: 255) GGAG-3′ (SEQ ID NO: 256) Number 60 M22D113-RM22D113-F D113A 5′-TCCCCAGCGTAGTAGAAATGCTGGGAGCT5′-CTACTACGCTGGGGAGCTCTTCCTCTCCCA CCTGGTGC-3′ (SEQ ID NO: 257)AAACCTG-3′ (SEQ ID NO: 258) Number 61 M22E115-R M22E115-F E115A5′-GCCCCATCGTAGTAGAAATGCTGGGAGCT 5′-CTACTACGATGGGGCGCTCTTCCTCTCCCACCTGGTGC-3′ (SEQ ID NO: 259) AAACCTG-3′ (SEQ ID NO: 260)

Antibodies did not show any loss of binding to unmutated wild type MICAbut lost binding to one or more mutants, thereby identifying severalepitopes.

Antibody 6E4 had loss of binding to mutants 10 and 11 having Q48A, W49S,E51S, D52A, V53S and L54A substitutions, but did not lose binding to anyother mutants. The principal epitope of 6E4 therefore includes one ormore of residues Q48 and W49, and/or one or more of residues E51, D52,V53 and L54. These residues are within the α1 domain of MICA (theepitope may further include residues within the α2 or α3 domains).

FIG. 2A shows a view of the MICA polypeptide, including in dark shadingthe amino acid residues mutated which resulted (in differentcombinations) in loss of binding by antibodies. The NKG2D binding siteis shown at the top of the MICA polypeptide in medium shading (and alsoin ribbon diagram bound to MICA). It can be seen that 6E4 binds to theα1 domain at the lateral side of MICA away from the NKG2D bindingsurface, consistent with the finding that 6E4 does not block MICA-NKG2Dinteractions.

Antibodies 9C10 and 12A10 had loss of binding to mutants 12 and 13having N56A, K57S, T58A, R61A and R64A substitutions, but did not losebinding to any other mutants. The principal epitope of 9C10 and 12A10therefore includes one or more of residues N56 and K57, and/or one ormore of residues T58, R61, and R64. These residues are within the α1domain of MICA (the epitope may further include residues within the α2or α3 domains).

FIG. 2B shows a view of the MICA polypeptide, including in dark shadingthe amino acid residues mutated which resulted (in differentcombinations) in loss of binding by 9C10 and 12A10. It can be seen that9C10 and 12A10 bind to the α1 domain at the lateral side of MICA nearthe NKG2D binding surface with possible partial overlap, consistent withthe finding that 9C10 and 12A10 block MICA-NKG2D interactions.

Antibody 20C6 had loss of binding to each of mutants 16, 17, 21, 60, 27and 28 having K81A D82A Q83A K84A H109A Y111A D113A L116A S133A R134ST137A M140S N141A R143S N144A substitutions, but did not lose binding toany other mutants. The principal epitope of 20C6 therefore includes oneor more of residues K81 and D82, one or more of residues Q83 and K84,one or more of residues H109, Y111 and L116, residue D113, one or moreof residues S133, R134 and T137, and/or one or more of residues M140,N141, R143 and N144. These residues are within the α1 and α2 domains ofMICA (the epitope may further include residues within the α3 domains).

Antibody 10A7 had partial overlap of epitope with 20C6. 10A7 had loss ofbinding to each of mutants 16, 17, 21, 60, 26 and 28 having K81A, D82A,Q83A, K84A, H109A, Y111A, D113A, L116A, Q131A, S132A, Q136S, M140S,N141A, R143S and N144A substitutions, but did not lose binding to anyother mutants. The principal epitope of 10A7 therefore includes one ormore of residues K81 and D82, one or more of residues Q83 and K84, oneor more of residues H109, Y111 abd L116, residue D113, one or more ofresidues Q131, S132 and Q136, and/or one or more of residues M140, N141,R143 and N144. These residues are within the α1 and α2 domains of MICA(the epitope may further include residues within the α3 domains).

FIG. 2C shows a view of the MICA polypeptide, including in dark shadingthe amino acid residues mutated which resulted (in differentcombinations) in loss of binding by 20C6. FIG. 2D shows a view of theMICA polypeptide, including in dark shading the amino acid residuesmutated which resulted (in different combinations) in loss of binding by10A7. It can be seen that 20C6 and 10A7 bind to the α1 and (32 domainsat the lateral side of MICA near the NKG2D binding surface with possiblepartial overlap. This is consistent with the finding that 20C6 blocksMICA NKG2D interactions.

Antibodies 19E9, 18E8 and 10F3 had loss of binding to mutants 19, 20, 23and 24 having E100A, D101S, N102A, S103A, T104S, R105A, N121A, E123S,T124A and E126A substitutions, but did not lose binding to any othermutants. The principal epitope of 19E9, 18E8 and 10F3 therefore includesone or more of residues E100, D101 and N102, one or more of residuesS103, T104, and R105, one or more of residues N121, and E123, and/or oneor more of residues T124 and E126. These residues are within the α2domain of MICA (the epitope may further include residues within the α1or α3 domains).

FIG. 2E shows a view of the MICA polypeptide, including in dark shadingthe amino acid residues mutated which resulted (in differentcombinations) in loss of binding by 19E9, 18E8 and 10F3. It can be seenthat 19E9, 18E8 and 10F3 bind to the α2 domain at the lateral side ofMICA near the NKG2D binding surface, consistent with the finding that19E9, 18E8 and 10F3 block MICA-NKG2D interactions.

Antibody 15F9 had loss of binding to mutants 1, 18, 19, 20, 61 and 36,having R6A, N8A, E97A, H99A, E100A, D101S, N102A, S103A, T104S, R105A,E115A, L178A, R179S and R180A substitutions, but did not lose binding toany other mutants. The principal epitope of 15F9 therefore includes oneor more of residues R6 and N8, one or more of residues E97 and H99, oneor more of residues E100, D101 and N102, one or more of residues S103,T104 and R105, residue E115, and/or one or more of residues L178, R179and R180. These residues are within the α2 domain of MICA (the epitopemay further include residues within the α1 or α3 domains).

FIG. 2F shows a view of the MICA polypeptide, including in dark shadingthe amino acid residues mutated which resulted (in differentcombinations) in loss of binding by 15F9. It can be seen that 15F9 bindsto the α2 domain at the lateral side of MICA below the NKG2D bindingsurface. 15F9 blocks sMICA-NKG2D interactions.

Antibody 16A8 had loss of binding to mutants, 45, 46, and 47, having,S224A, H225S, D226A, T227A, Q228S, Q229A, W230A and D232A substitutions,but did not lose binding to any other mutants. The principal epitope of16A8 therefore including one or more of residues W230 and/or D232, oneor more of residues T227, Q228 and Q229, one or more of residues S224,H225 and D226. The epitope of 16A8 is primarily within the α3 domain ofMICA.

FIG. 2G shows a view of the MICA polypeptide, including in dark shadingthe amino acid residues mutated which resulted (in differentcombinations) in loss of binding by 16A8. It can be seen that 16A8 bindsto the α3 domain away from the NKG2D binding surface.

Antibody 14B4 had loss of binding to mutant 46, having T227A, Q228S, andQ229A substitutions, but did not lose binding to any other mutants. Theprincipal epitope of 14B4 therefore includes one or more of residuesT227, Q228 and Q229, and had a partial overlap with antibody 16A8. Theepitope of 14B4 is primarily within the α3 domain of MICA. It can beseen that 1464 binds to the α3 domain away from the NKG2D bindingsurface (however 14B4 is a functionally blocking antibody).

Example 5—Ability of Anti-MICA Antibodies to Block NKG2D-MICAInteractions

A. Binding of MICA*001-his to NKG2D-Fc in Presence of Anti-MICAAntibodies Assessed by Surface Plasmon Resonance

SPR measurements were performed on a Biacore T100 apparatus (Biacore GEHealthcare) at 25° C. In all Biacore experiments HBS-EP+ buffer (BiacoreGE Healthcare) served as running buffer, 10 mM NaOH, 500 mM NaCl servedas regeneration buffer and sensorgrams were analyzed with Biaevaluation4.1 and Biacore T100 Evaluation software.

For solution competition experiments, the human NKG2D-Fc (R&D systems)recombinant proteins were covalently immobilized onto a CM5 sensorchip.Soluble human MICA*01-BirA or human MICA*019-Fc (R&D systems)recombinant proteins at a constant concentration of 10 μg/ml werepre-incubated with a 5 to 10 molar equivalent excess of antibodies andinjected for 2 minutes at a flow rate of 10 μl/min onto the NKG2D-Fcchip. After each cycle, Sensorchips were regenerated by a five secondinjection of appropriate regeneration buffer. FIG. 3 shows arepresentative example of results and results are summarized in Table E

B. Ability of Anti-MICA Antibodies to Block NKG2D-Dependent Lysis ofRaji-MICA*08 Transfectant Cells by the NK92 NK Cell Line.

The ability of anti-MICA antibodies to block the NKG2D-MICA interactionwas assessed. Antibodies were tested for the ability to reduce orinhibit the NKG2D+CD16-NK92 cell-mediated killing ofMICA*008-transfected Raji by measuring target cell release of ⁵¹Cr. Thein vitro cytotoxicity assay was carried out by standard methods that arewell known in the art, as described for example in Coligan et al., eds.,Current Protocols in Immunology, Greene Publishing Assoc. and WileyInterscience, N.Y., (1992, 1993). The MICA-expressing target cells werelabeled with ⁵¹Cr prior to addition of NK cell line, and then thekilling was estimated as proportional to the release of ⁵¹Cr from thecells to the medium, as a result of killing. Addition of an agent thatreduces binding or blocks an interaction between MICA and NKG2D resultedin prevention of the initiation and propagation of activatory signallingvia NKG2D. Therefore addition of such agents results in decreases inNK-mediated killing of the target cells. F(ab′)2 fragment of thecommercially available blocking anti-NKG2D antibody is used as apositive control of NKG2D-MICA blocking, Rituximab (chimeric human IgG1anti-CD20) is used as a negative control to ensure that lysis is notmediated through ADCC and an additional negative control in the form ofan irrelevant chimeric human IgG1 produced in the same conditions as theanti-MICA is used. Examples of results are shown in FIG. 4 andsummarized in Table E.

The CHG1-M-MIA-20C6, CHG1-M-MIA-10A7 do block the interaction ofrecombinant non-glycosylated MICA*001-BirA with bivalent NKG2D-Fcrecombinant protein whereas they do not block the NKG2D-mediated killingof Raji-MICA*08 by NK92. The first hypothesis is that these twoanti-MICA have the lowest monovalent affinities for recombinantMICA*001, thus resulting in a higher amount of antibody than testedrequired to block the NKG2D-MICA interaction in a cell-to-cell cytotoxicassay. Interestingly, this would imply that when used in vivo in thetreatment of a tumor, the antibodies might have some blocking ability athigh dose (i.e. at a time when ADCC/CDC is the predominant activemechanism because the mAb concentration is sufficient to causesignificant depletion of MICA+ cells by ADCC/CDC), but that as theantibody concentration in vivo decreases in the days/weeks afteradministration of a (high) dose, the antibodies will becomenon-blocking, permitting patients' endogenous NKG2D receptors tofunction optimally. Alternatively, the blocking capacity assessed usingrecombinant proteins in Biacore may not be predictive of the complexityof the molecular NKG2D-MICA interaction between native fullyglycosylated proteins presented at the cell membrane. Both NKG2D-Fc andMICA recombinant proteins may not be in their native conformation duringthe test. Finally, there may exist some discrete alleles specificitiesin term of quaternary structure that could be responsible for thedifferences in blocking capacity observed between the two experimentalprocedures used.

In addition, epitopes are not predictive of the NKG2D-MICA blockingcapacity as CHG1-M-MIA-19E9 for example is a blocking anti-MICA antibodyin both assay with an epitope not directly on the NKG2D binding site(FIG. 2E). CHG1-M-MIA-14B4, when binding to tested allele, is blockingNKG2D-MICA interaction although its epitope is on the α3 domainsuggesting that MICA conformation is altered upon CHG1-M-MIA-14B4binding. The epitope for CHG1-M-MIA-20C6 is near the NKG2D binding sitebut not overlapping (FIGS. 2C, 2D and 2F) and appears to be notfunctionally blocking on MICA*008.

TABLE E Summary of NKG2D-MICA blocking capacity of chimeric anti-MICAantibodies assessed by surface plasmon resonance or by a functionalcytotoxicity assay NKG2D-Fc/MICA*001- NKG2D-Fc/MICA*019- NK92/Raji- BirAinteraction Fc interaction MICA*08 lysis (Surface Plasmon (SurfacePlasmon (Cytotoxicity Resonance) Resonance) Assay) CHG1-M-MIA-9C10Blocking Blocking Blocking CHG1-M-MIA-12A10 Blocking BlockingCHG1-M-MIA-19E9 Blocking Blocking Blocking CHG1-M-MIA-18E8 BlockingBlocking CHG1-M-MIA-10F3 Blocking Blocking CHG1-M-MIA-15F9 Non BlockingNon Blocking CHG1-M-MIA-6E4 Non Blocking Non Blocking Non BlockingCHG1-M-MIA-20C6 Blocking Blocking Non Blocking CHG1-M-MIA-10A7 BlockingNon Blocking CHG1-M-MIA-16A8 Non Blocking Not Binding to Non BlockingMICA*019-Fc CHG1-M-MIA-14B4 Not binding to Blocking BlockingMICA*001-BirA

Example 6—Crossreaction of Anti-MICA Antibodies with Human MICB, ULBP1,ULBP2 and ULBP3

For cross-binding study, the human ULBP-1-Fc (R&D systems*), MICB-Fc(*), ULBP-2-Fc (*) and ULBP-3-Fc (*) recombinant proteins werecovalently immobilized respectively on the flow cell one to four of aCM5 sensorchip. Anti-MICA antibodies (at a constant concentration of 10μg/ml) were injected for 2 minutes at a flow rate of 10 μl/min onto thefour flow cells in parallel. After each cycle, Sensorchip wasregenerated by a five second injection of appropriate regenerationbuffer.

FIG. 5A shows absence of crossreaction to MICB and ULPB-1, -2 and -3 ofCHG1-M-MIA-20C6 and CHG1-M-MIA-9010. FIG. 5B shows that CHG1-M-MIA-6E4and CHG1-M-MIA-19E9 bind to MICB but not to ULBP-1, -2 and -3. FIG. 5Cshows that CHG1-M-MIA-16A8 binds weakly but significantly to MICB butnot to ULBP-1, -2 and -3. Antibodies that were not tested are expectedto have similar profiles as other antibodies that share same epitoperegions.

Table F summarizes results with all tested anti-MICA antibodies.

TABLE F Crossreaction of chimeric anti-MICA antibodies to MICB, ULBP-1,ILBP-2 and ULBP-3 assessed by surface plasmon resonance. CrossreactionCrossreaction Crossreaction Crossreaction on human on human on human onhuman MICB-Fc ULBP1-Fc ULBP2-Fc ULBP3-Fc CHG1-M-MIA-9C10 No No No NoCHG1-M-MIA-12A10 No Not Tested Not Tested Not Tested CHG1-M-MIA-19E9 YesNo No No CHG1-M-MIA-18E8 Yes Not Tested Not Tested Not TestedCHG1-M-MIA-10F3 Yes Not Tested Not Tested Not Tested CHG1-M-MIA-15F9 YesNot Tested Not Tested Not Tested CHG1-M-MIA-6E4 Yes No No NoCHG1-M-MIA-20C6 No No No No CHG1-M-MIA-10A7 No Not Tested Not Tested NotTested CHG1-M-MIA-16A8 Yes (low) No No No CHG1-M-MIA-14B4 No Not TestedNot Tested Not Tested

Example 7—Crossreaction of Anti-MICA Antibodies with Macaca fascicularisMIC Proteins

The binding of antibodies obtained from the first, second and thirdimmunization series (including CHG1-M-MIA-19E9, CHG1-M-MIA-9C10,CHG1-M-MIA-6E4, CHG1-M-MIA-20C6, CHG1-M-MIA-16A8, CHG1-M-MIA-12A10,CHG1-M-MIA-18E8, CHG1-M-MIA-10F3, CHG1-M-MIA-15F9 and CHG1-M-MIA-14B4)were tested for binding to Macaca fascicularis (Cynomolgus) MIC homologsproteins. 3 sequences were successfully cloned from Macaca fasciculariscDNA including MIC #9-1, MIC #9-2 and MIC #2-7 and transfected in themouse cell line Baf/3. Binding was analyzed by flow cytometry, NKG2D-Fcrecombinant protein is used as a control to detect surface expression ofMacaca fascicularis MIC proteins.

Flow Cytometry.

Cells were harvested and stained in PBS 1×/BSA 0.2%/EDTA 2 mM bufferduring 30 minutes at 4° C. using a dose-range of the anti-MICA mAbs orwith NKG2D-Fc recombinant protein (R&D systems). After two washes instaining buffer, cells incubated with anti-MICA antibodies were stainedfor 30 min at 4° C. with mouse anti-human IgG1-PE monoclonal antibodies(1/11). Cells incubated with NKG2D-Fc were stained for 30 min at 4° C.with goat anti-human IgG polyclonal antibodies (1/200). After twowashes, stainings were acquired on a BD FACS Canto II and analyzed usingthe BD FACSDiva software.

CHG1-M-MIA-19E9, CHG1-M-MIA-9C10, CHG1-M-MIA-6E4, CHG1-M-MIA-20C6,CHG1-M-MIA-16A8, CHG1-M-MIA-12A10, CHG1-M-MIA-10F3, CHG1-M-MIA-15F9 andCHG1-M-MIA-14B4 antibodies bind to MIC #2-7 Macaca fascicularis protein.MIC #2-7 presents 99.6%, 97.8%, 95.6% and 86.9% protein homology withMICA*019, MICA*044, MICA*001 and MICB*002 respectively. In addition toMIC #2-7, CHG1-M-MIA-6E4 also binds to MIC #9-1. CHG1-M-MIA-16A8 bindsto MIC #9-2 protein in the experimental conditions tested. Results areshown in FIG. 6.

Example 8—Inhibition of MICA Shedding

Anti-α3 domain 16A8 antibody was compared to commercially availableBAMO3 (see Salih et al (2003), supra) for its capacity to block MICAshedding. A mix of C1R-MICA*01 and C1R-MICA*04 cells were washed in PBS1× to remove soluble MICA present in the culture and then culturedovernight in complete medium in the presence or absence of a dose range(0/1/3/10/30 μg/ml) of 16A8 or BAM03. Then cell culture supernatantswere harvested and tested in ELISA for the presence of soluble MICA.Neither 16A8 nor BAMO3 are interfering with the anti-MICA antibodiesused in the ELISA (data not shown). Overnight incubation of the cellswith BAMO3 results in a decrease of the soluble MICA concentration inthe supernatant whereas 16A8 does not induce a decrease of the solubleMICA. Results are shown in FIG. 7, showing an inhibition of the MICAshedding mediated by BAMO3 but not by 16A8.

Example 9—Effect of Anti-MICA Antibodies on NKG2D Downmodulation

Reports have emerged that NKG2D on NK cells is downregulated by sMICA(Groh et al. (2002) Nature; Arreygue-Garcia (2008) BMC; Jinushi et al.(2005) J. Hepatol.), leading to less reactive NK cells. To mimicdownregulation induced by soluble MICA the following experiments wereperformed.

Thawed PBMC from four healthy donors were enriched in lymphocytesfollowing non-specific removal of monocytes by cold-aggregation(Rubinstien (1989) J. Clin. Lab. Immunol.). Cells (1.10e5 cells perwells) were incubated in a 96-well plate in the presence of increasingconcentrations of soluble MICA*019-Fc (R&D systems) in the presence orabsence of anti-MICA antibodies (CHG1-M-MIA-9C10, CHG1-M-MIA-19E9,CHG1-M-MIA-20C6 at 10 μg/mL) for 24h at 4° C. or 37° C. NKG2Ddownmodulation induced by soluble MICA and its blockade by anti-MICAantibodies was assessed by flow cytometry. Cells were stained with thefollowing human-specific mouse antibodies: anti-CD14 FITC, anti-CD3Pacific blue (Becton Dickinson), anti-CD56 PE-Cy7 (BioLegend),anti-NKG2D PE (Beckman Coulter) and analyzed for NKG2D expression ongated CD3-CD56+NK cells.

In this experimental setting, NKG2D is downmodulated by 30 to 40% of itsinitial level in presence of increasing doses of recombinant bivalentMICA*019*Fc protein (R&D systems) (FIG. 8). This effect is seen between2 to 10 μg/ml in the culture medium, much above described levels ofsoluble MICA observed in sera from cancer patients (ranging 30 to 1557μg/ml in malignant disorders, n=296, Holdenrieder (2006) Int. J.Cancer). The NKG2D downmodulation is not observed in these experimentalconditions using monovalent MICA*01-His recombinant protein (data notshown). CHG1-M-MIA-9C10 and CHG1-M-MIA-19E9 anti-MICA antibodies,blocking NKG2D-MICA interaction in the cytotoxic assay of Example 5B(Table E), are blocking the interaction of NKG2D expressed on NK cellswith MICA*019-Fc, hence reversing the NKG2D downmodulation induced bythe MICA*019-Fc protein (FIG. 8). CHG1-M-MIA-20C6 anti-MICA does notreverse MICA*019-Fc-mediated NKG2D downmodulation (FIG. 8). Althoughthis antibody is blocking NKG2D-Fc/MICA*019-Fc binding by surfaceplasmon resonance (Table E), it is not blocking NKG2D/MICA*08 in thecellular cytotoxic assay of Example 5B (Table E) and is not reversingthe NKG2D downmodulation on primary cells emphasizing the fact thatNKG2D/MICA blocking experiments using recombinant proteins may not bepredictive of the biological function, at least in the experimentalconditions tested.

Example 10—Anti-MICA Antibodies are Able to Mediate Killing of MICAExpressing Targets Via CDC

CHG1-M-MIA-9C10, CHG1-M-MIA-19E9, CHG1-M-MIA-6E4, CHG1-M-MIA-20C6 andCHG1-M-MIA-16A8, were tested for their ability to mediate CDC towardshuman Raji tumor cells transduced with a lentivirus encoding forMICA*001 full protein. These Raji-MICA*01 cells express MICA*01 at theircell surface.

Briefly, 100 000 Raji-MICA*01 cells were incubated 1h with the indicateddoses of anti-MICA antibodies at 4° C. Then, culture medium containing20% (final concentration) of Human Serum Complement (Quidel) was addedto the cells and incubated at 37° C. for 3 hours. Cells were washed andincubated with 7-AAD to stain dead cells. Cells were acquired by flowcytometry on a BD FACS Canto II and analyzed using the BD FACSDivasoftware. Results are expressed as a percentage of 7-AAD positive cellsin the indicated condition.

Results are shown in FIG. 9. The results show viability of indicatedRaji-MICA*01 cells, in the presence of human complement. The resultsshow that CHG1-M-MIA-20C6, CHG1-M-MIA-9C10, CHG1-M-MIA-6E4, andCHG1-M-MIA-19E9 cause an increase the number of dead cells (with EC50values of 0.97, 2, 1.01 and 2.35 μg/ml respectively) (FIG. 9). Themaximum percentage of dead cells varies with the different anti-MICAtested (FIG. 9). Notably, CHG1-M-MIA-16A8 is not mediating complementcytotoxicity. As these 5 anti-MICA have EC50 values for stainingC1R-MICA*01 of the same order (Table C), complement-mediatedcytotoxicity appears to be epitope-dependent (9C10>20C6=6E4>19E9»16A8).

Example 11—Antibodies are Able to Kill MICA Expressing Targets Via ADCC

CHG1-M-MIA-19E9, CHG1-M-MIA-9C10, CHG1-M-MIA-6E4, CHG1-M-MIA-20C6,CHG1-M-MIA-16A8, CHG1-M-MIA-12A10, CHG1-M-MIA-18E8, CHG1-M-MIA-10F3,CHG1-M-MIA-15F9 and CHG1-M-MIA-14B4 were tested for their ability tomediate ADCC towards C1R tumor cells transfected with MICA*008(C1R-MICA*008).

Briefly, the cytolytic activity of human NK cell line KHYG-1 transfectedwith human CD16 (V isoform) was assessed in a classical 4-h ³¹Cr-releaseassay in 96 well plates V from (Greiner). Briefly, C1R-MICA*008 cellswere labelled with ⁵¹Cr (100 μCi (3.7 MBq)/1×10⁶ cells), then mixed withKHYG-transfected with hCD16V (to bind human IgG1) at an effector/targetratio equal to 20, in the presence of antibody at indicatedconcentrations and of 10 mg/ml F(ab′)2 ON-72 to block any NKG2D-mediatedcytotoxicity). After brief centrifugation and 4 hours of incubation at37° C., 50 μL supernatant were removed, and the ⁵¹Cr release wasmeasured with a TopCount NXT beta detector (PerkinElmer Life Sciences,Boston, Mass.). All experimental groups were analyzed in triplicate, andthe percentage of specific lysis was determined as follows: 100×(meancpm experimental release−mean cpm spontaneous release)/(mean cpm totalrelease−mean cpm spontaneous release). Percentage of total releaseobtained by lysis of target cells with 2% Triton X100 (Sigma).

Results are shown in FIG. 10A. CHG1-M-MIA-9C10, CHG1-M-MIA-19E9,CHG1-M-MIA-6E4, CHG1-M-MIA-20C6 and CHG1-M-MIA-16A8 each inducedspecific lysis of C1R-MICA*008 cells by human KHYG-1 hCD16V NK cellsline compared to negative controls (Human IgG1 isotype controlantibody), thereby showing that these antibodies induce ADCC towardMICA*008-expressing target cells. The extent of target cell lysis iscorrelated to antibody binding to the cell (FIG. 10B).

Example 12—Chimeric Anti-MICA 9C10, 19E9, 6E4, 20C6 and 16A8 ShowAnti-Tumoral Efficacy in a Mouse Model of RAJI-MICA*01High Xenograft

Antibodies were tested in a mouse long-term RAJI-MICA*01 tumor model inwhich RAJI cells expressed high level of antigen MICA*01. Nod SCID micewere intravenously (IV) engrafted with 15.10⁶ RAJI-MICA*01High andtreated with either control isotype control antibody (IC) or chimericantibodies CHG1-M-MIA-9C10, CHG1-M-MIA-19E9, CHG1-M-MIA-6E4,CHG1-M-MIA-20C6, or CHG1-M-MIA-16A8 at the dosage of 300 μg/mouse, IPtwice/week for 3 weeks from the day of tumor cell graft.

RAJI-MICA*01High were cultured in complete RPMI 1640 culture mediumcontaining supplemented with 10% of Fetal Bovine Serum Heat Inactivated,1% L-glutamine, 1% Sodium/Pyruvate and without antibodies prior toinjection into mice.

Mice were weighed twice per week. Kaplan-Meier survival curves wereestablished to assess survival of treated mice.

Results are shown in FIG. 11. All chimeric antibodies showedanti-tumoral activity. Animals receiving isotype control had a mediansurvival of 27 days whereas mice treated with the least effectivechimeric antibody (16A8) had a median survival of 77 days. For all otherantibodies, more than 50% of animals are still alive at day 100,preventing the calculation of the median survival and indicating a verystrong anti-tumoral effect.

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way. Anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context. Recitation ofranges of values herein are merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recitedherein. Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e. g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability and/or enforceability of such patent documents, Thedescription herein of any aspect or embodiment of the invention usingterms such as reference to an element or elements is intended to providesupport for a similar aspect or embodiment of the invention that“consists of”, “consists essentially of” or “substantially comprises”that particular element or elements, unless otherwise stated or clearlycontradicted by context (e. g., a composition described herein ascomprising a particular element should be understood as also describinga composition consisting of that element, unless otherwise stated orclearly contradicted by context).

This invention includes all modifications and equivalents of the subjectmatter recited in the aspects or claims presented herein to the maximumextent permitted by applicable law.

All publications and patent applications cited in this specification areherein incorporated by reference in their entireties as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1-47. (canceled)
 48. A monoclonal antibody comprising (i) a heavy chaincomprising the CDR 1 (SEQ ID NO: 92), 2 (SEQ ID NO: 95) and 3 (SEQ IDNO: 97) of the heavy chain variable region of SEQ ID NO: 90 and (ii) alight chain comprising the CDR 1 (SEQ ID NO: 98), 2 (SEQ ID NO: 99) and3 (SEQ ID NO: 100) of the light chain variable region of SEQ ID NO: 91.49. The antibody of claim 48, wherein said antibody binds to a cellsurface bound MICA polypeptide comprising an amino acid sequence of SEQID NO: 1, a cell surface bound MICA polypeptide comprising an amino acidsequence of SEQ ID NO: 2, and a cell surface bound MICA polypeptidecomprising an amino acid sequence of SEQ ID NO:
 4. 50. The antibody ofclaim 48, wherein said antibody has reduced binding to a mutant MICApolypeptide comprising a mutation at 1, 2, 3, 4 or more residuesselected from the group consisting of E100, D101, N102, S103, T104,R105, N121, E123, T124 and E126, relative to binding between theantibody and a wild-type MICA polypeptide of SEQ ID NO:
 1. 51. Apharmaceutical composition comprising an antibody of claim 48, and apharmaceutically acceptable carrier.
 52. The antibody of claim 48,wherein said antibody is coupled to a cytotoxic agent.
 53. The antibodyof claim 48, wherein said antibody is conjugated or coupled to adetectable agent.
 54. A recombinant nucleic acid encoding a heavy and/orlight chain of the antibody of claim
 48. 55. A hybridoma or recombinanthost cell producing the antibody of claim
 48. 56. A method for thetreatment of a cancer in a patient in need thereof, the methodcomprising administering to said patient an effective amount acomposition of claim
 51. 57. A method for identifying a MICA-expressingcell in a subject, the method comprising obtaining a biological samplefrom a subject comprising cells, bringing said cells into contact withan antibody of claim 48 and assessing whether the antibody binds to thecells.
 58. The method of claim 57, wherein said method is free of anadditional prior step of determining whether said subject or cellscomprise a MICA allele selected from the group consisting of MICA*001,MICA*004, MICA*007 and MICA*008.
 59. A method for delivering a toxicmolecule to a tumor cell, the method comprising exposing a tumor cellwhich expresses a MICA polypeptide to a monoclonal antibody of claim 48linked to a toxic agent.